Method for the production of 1-butanol

ABSTRACT

A method for the production of 1-butanol by fermentation using a microbial production host is disclosed. The method employs a reduction in temperature during the fermentation process that results in a more robust tolerance of the production host to the butanol product.

FIELD OF THE INVENTION

The invention relates to a method for the production of 1-butanol byfermentation using a recombinant microbial host. Specifically, themethod employs a decrease in temperature during fermentation thatresults in more robust tolerance of the production host to the 1-butanolproduct.

BACKGROUND OF THE INVENTION

Butanol is an important industrial chemical, useful as a fuel additive,as a feedstock chemical in the plastics industry, and as a foodgradeextractant in the food and flavor industry. Each year 10 to 12 billionpounds of butanol are produced by petrochemical means and the need forthis commodity chemical will likely increase.

Methods for the chemical synthesis of 1-butanol are known, such as theOxo Process, the Reppe Process, and the hydrogenation of crotonaldehyde(Ullmann's Encyclopedia of Industrial Chemistry, 6^(th) edition, 2003,Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719).These processes use starting materials derived from petrochemicals andare generally expensive and are not environmentally friendly. Theproduction of 1-butanol from plant-derived raw materials would minimizegreenhouse gas emissions and would represent an advance in the art.

Methods of producing 1-butanol by fermentation are also known, where themost popular process produces a mixture of acetone, 1-butanol andethanol and is referred to as the ABE process (Blaschek et al., U.S.Pat. No. 6,358,717). Acetone-butanol-ethanol (ABE) fermentation byClostridium acetobutylicum is one of the oldest known industrialfermentations, and the pathways and genes responsible for the productionof these solvents have been reported (Girbal et al., Trends inBiotechnology 16:11-16 (1998)). Additionally, recombinant microbialproduction hosts expressing a 1-butanol biosynthetic pathway have beendescribed (Donaldson et al., copending and commonly owned U.S. patentapplication Ser. No. 11/527,995). However, biological production of1-butanol is believed to be limited by butanol toxicity to the hostmicroorganism used in the fermentation.

Some microbial strains that are tolerant to 1-butanol are known in theart (see for example, Jain et al. U.S. Pat. No. 5,192,673; Blaschek etal. U.S. Pat. No. 6,358,717; Papoutsakis et al. U.S. Pat. No. 6,960,465;and Bramucci et al., copending and commonly owned U.S. patentapplication Ser. Nos. 11/743,220, 11/761,497, and 11/949,793). However,biological methods of producing 1-butanol to higher levels are requiredfor cost effective commercial production.

There have been reports describing the effect of temperature on thetolerance of some microbial strains to ethanol. For example, Amartey etal. (Biotechnol. Lett. 13(9):627-632 (1991)) disclose that Bacillusstearothermophillus is less tolerant to ethanol at 70° C. than at 60° C.Herrero et al. (Appl. Environ. Microbiol. 40(3):571-577 (1980)) reportthat the optimum growth temperature of a wild-type strain of Clostridiumthermocellum decreases as the concentration of ethanol challengeincreases, whereas the optimum growth temperature of an ethanol-tolerantmutant remains constant. Brown et al. (Biotechnol. Lett. 4(4):269-274(1982)) disclose that the yeast Saccharomyces uvarum is more resistantto growth inhibition by ethanol at temperatures 5° C. and 10° C. belowits growth optimum of 35° C. However, fermentation became more resistantto ethanol inhibition with increasing temperature. Additionally, VanUden (CRC Crit. Rev. Biotechnol. 1(3):263-273 (1984)) report thatethanol and other alkanols depress the maximum and the optimum growthtemperature for growth of Saccharomyces cerevisiae while thermal deathis enhanced. Moreover, Lewis et al. (U.S. patent Application PublicationNo. 2004/0234649) describe methods for producing high levels of ethanolduring fermentation of plant material comprising decreasing thetemperature during saccharifying, fermenting, or simultaneouslysaccharifying and fermenting.

Much less is known about the effect of temperature on the tolerance ofmicrobial strains to 1-butanol. Harada (Hakko Kyokaishi 20:155-156(1962)) discloses that the yield of 1-butanol in the ABE process isincreased from 18.4%-18.7% to 19.1%-21.2% by lowering the temperaturefrom 30° C. to 28° C. when the growth of the bacteria reaches a maximum.Jones et al. (Microbiol. Rev. 50(4):484-524 (1986)) review the role oftemperature in ABE fermentation. They report that the solvent yields ofthree different solvent producing strains remains fairly constant at 31%at 30° C. and 33° C., but decreases to 23 to 25% at 37° C. Similarresults were reported for Clostridium acetobutylicum for which solventyields decreased from 29% at 25° C. to 24% at 40° C. In the latter case,the decrease in solvent yield was attributed to a decrease in acetoneproduction while the yield of 1-butanol was unaffected. However,Carnarius (U.S. Pat. No. 2,198,104) reports that an increase in thebutanol ratio is obtained in the ABE process by decreasing thetemperature of the fermentation from 30° C. to 24° C. after 16 hours.However, the effect of temperature on the production of 1-butanol byrecombinant microbial hosts is not known in the art.

There is a need, therefore, for a cost-effective process for theproduction of 1-butanol by fermentation that provides higher yields thanprocesses known in the art. The present invention addresses this needthrough the discovery of a method for producing 1-butanol byfermentation using a recombinant microbial host, which employs adecrease in temperature during fermentation, resulting in more robusttolerance of the production host to the 1-butanol product.

SUMMARY OF THE INVENTION

The invention provides a method for the production of 1-butanol byfermentation using a recombinant microbial host, which employs adecrease in temperature during fermentation that results in more robusttolerance of the production host to the 1-butanol product.

Accordingly, the invention provides a method for the production of1-butanol comprising:

-   -   a) providing a recombinant microbial production host which        produces 1-butanol;    -   b) seeding the production host of (a) into a fermentation        -   medium comprising a fermentable carbon substrate to create a            fermentation culture;    -   c) growing the production host in the fermentation culture at a        first temperature for a first period of time;    -   d) lowering the temperature of the fermentation culture to a        second temperature; and    -   e) incubating the production host at the second temperature of        step (d) for a second period of time;    -   whereby 1-butanol is produced.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription, FIGURE, and the accompanying sequence descriptions, whichform a part of this application.

FIG. 1 shows the 1-butanol biosynthetic pathway. The steps labeled “a”,“b”, “c”, “d”, “e”, and “f” represent the substrate to productconversions described below.

The following sequences conform with 37 C.F.R. 1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

TABLE 1 Summary of Gene and Protein SEQ ID Numbers SEQ ID NO SEQ NucleicID NO Description acid Peptide Acetyl-CoA acetyltransferase thlA 1 2from Clostridium acetobutylicum ATCC 824 Acetyl-CoA acetyltransferasethlB 3 4 from Clostridium acetobutylicum ATCC 824 Acetyl-CoAacetyltransferase from 128 129 Escherichia coli Acetyl-CoAacetyltransferase from 130 131 Bacillus subtilis Acetyl-CoAacetyltransferase from 132 133 Saccharomyces cerevisiae3-Hydroxybutyryl-CoA 5 6 dehydrogenase from Clostridium acetobutylicumATCC 824 3-Hydroxybutyryl-CoA 134 135 dehydrogenase from Bacillussubtilis 3-Hydroxybutyryl-CoA 136 137 dehydrogenase from Ralstoniaeutropha 3-Hydroxybutyryl-CoA 138 139 dehydrogenase from Alcaligeneseutrophus Crotonase from Clostridium 7 8 acetobutylicum ATCC 824Crotonase from Escherichia coli 140 141 Crotonase from Bacillus subtilis142 143 Crotonase from Aeromonas caviae 144 145 Putative trans-enoyl CoAreductase 9 10 from Clostridium acetobutylicum ATCC 824 Butyryl-CoAdehydrogenase from 146 147 Euglena gracilis Butyryl-CoA dehydrogenasefrom 148 149 Streptomyces collinus Butyryl-CoA dehydrogenase from 150151 Streptomyces coelocolor Butyraldehyde dehydrogenase from 11 12Clostridium beijerinckii NRRL B594 Butyraldehyde dehydrogenase from 152153 Clostridium acetobutylicum Butanol dehydrogenase bdhB from 13 14Clostridium acetobutylicum ATCC 824 Butanol dehydrogenase 15 16 bdhAfrom Clostridium acetobutylicum ATCC 824 Butanol dehydrogenase 152 153from Clostridium acetobutylicum Butanol dehydrogenase 154 155 fromEscherichia coli

SEQ ID NOs:17-44 are the nucleotide sequences of oligonucleotide primersused to amplify the genes of the 1-butanol biosynthetic pathway.

SEQ ID NOs:45-72 are the nucleotide sequences of oligonucleotide primersused for sequencing.

SEQ ID NOs:73-75 are the nucleotide sequences of oligonucleotide primersused to construct the transformation vectors described in Example 13.

SEQ ID NO:76 is the nucleotide sequence of the codon-optimized CAC0462gene, referred to herein as CaTER.

SEQ ID NO:77 is the nucleotide sequence of the codon-optimized EgTERgene, referred to herein as EgTER(opt).

SEQ ID NO:78 is the nucleotide sequence of the codon-optimized ald gene,referred to herein as ald(opt).

SEQ ID NO:79 is the nucleotide sequence of the plasmid pFP988.

SEQ ID NO:'s 80-127, 160-185, and 190-207 are the nucleic acid sequencesof cloning, sequencing, or PCR screening primers used for the cloning,sequencing, or screening of the genes of the 1-butanol biosyntheticpathway described herein, and are more fully described in Tables 4 and5.

SEQ ID NO:156 is the nucleotide sequence of the cscBKA gene cluster.

SEQ ID NO:157 is the amino acid sequence of sucrose hydrolase (CscA).

SEQ ID NO:158 is the amino acid sequence of D-fructokinase (CscK).

SEQ ID NO:159 is the amino acid sequence of sucrose permease (CscB).

SEQ ID NO:186 is the nucleotide sequence of the codon optimized terygene described in Example 21.

SEQ ID NO:187 is the amino acid sequence of the butyl-CoA dehydrogenase(ter) encoded by the codon optimized tery gene (SEQ ID NO:186).

SEQ ID NO:188 is the nucleotide sequence of the codon optimized aldygene described in Example 21.

SEQ ID NO:189 is the amino acid sequence of the butyraldehydedehydrogenase (ald) encoded by the codon optimized aldy gene (SEQ IDNO:188).

SEQ ID NO:208 is the nucleotide sequence of the template DNA used inExample 18.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for the production of1-butanol using recombinant microorganisms that employs a decrease intemperature during fermentation, resulting in more robust tolerance ofthe production host to the 1-butanol product and therefore a highertiter of 1-butanol. The present invention meets a number of commercialand industrial needs. 1-Butanol is an important industrial commoditychemical with a variety of applications, where its potential as a fuelor fuel additive is particularly significant. Although only afour-carbon alcohol, butanol has an energy content similar to that ofgasoline and can be blended with any fossil fuel. Butanol is favored asa fuel or fuel additive as it yields only CO₂ and little or no SO_(X) orNO_(X) when burned in the standard internal combustion engine.Additionally 1-butanol is less corrosive than ethanol, the mostpreferred fuel additive to date.

In addition to its utility as a biofuel or fuel additive, 1-butanol hasthe potential of impacting hydrogen distribution problems in theemerging fuel cell industry. Fuel cells today are plagued by safetyconcerns associated with hydrogen transport and distribution. 1-Butanolcan be easily reformed for its hydrogen content and can be distributedthrough existing gas stations in the purity required for either fuelcells or vehicles.

Finally the present invention produces 1-butanol from plant derivedcarbon sources, avoiding the negative environmental impact associatedwith standard petrochemical processes for butanol production.

The following definitions and abbreviations are to be used for theinterpretation of the claims and the specification.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, are intended to cover a non-exclusive inclusion. Forexample, a composition, a mixture, process, method, article, orapparatus that comprises a list of elements is not necessarily limitedto only those elements but may include other elements not expresslylisted or inherent to such composition, mixture, process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive or and not to an exclusive or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the indefinite articles “a” and “an” preceding an element orcomponent of the invention are intended to be nonrestrictive regardingthe number of instances (i.e. occurrences) of the element or component.Therefore “a” or “an” should be read to include one or at least one, andthe singular word form of the element or component also includes theplural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdescribed in the specification and the claims.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates oruse solutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities. Inone embodiment, the term “about” means within 10% of the reportednumerical value, preferably within 5% of the reported numerical value.

“ABE” is the abbreviation for the Acetone-Butanol-Ethanol fermentationprocess.

The term “1-butanol biosynthetic pathway” means the enzyme pathway toproduce 1-butanol from acetyl-coenzyme A (acetyl-CoA).

The term “acetyl-CoA acetyltransferase” refers to an enzyme thatcatalyzes the conversion of two molecules of acetyl-CoA toacetoacetyl-CoA and coenzyme A (CoA). Preferred acetyl-CoAacetyltransferases are acetyl-CoA acetyltransferases with substratepreferences (reaction in the forward direction) for a short chainacyl-CoA and acetyl-CoA and are classified as E.C. 2.3.1.9 [EnzymeNomenclature 1992, Academic Press, San Diego]; although, enzymes with abroader substrate range (E.C. 2.3.1.16) will be functional as well.Acetyl-CoA acetyltransferases are available from a number of sources,for example, Escherichia coli (GenBank Nos: NP_(—)416728 (SEQ IDNO:129), NC_(—)000913 (SEQ ID NO:128); NCBI (National Center forBiotechnology Information) amino acid sequence, NCBI nucleotidesequence), Clostridium acetobutylicum (GenBank Nos: NP_(—)349476.1 (SEQID NO:2), NC_(—)003030 (SEQ ID NO:1); NP_(—)149242 (SEQ ID NO:4),NC_(—)001988 (SEQ ID NO:3), Bacillus subtilis (GenBank Nos: NP_(—)390297(SEQ ID NO:131), NC_(—)000964 (SEQ ID NO:130)), and Saccharomycescerevisiae (GenBank Nos: NP_(—)015297 (SEQ ID NO:133), NC_(—)001148 (SEQID NO:132)).

The term “3-hydroxybutyryl-CoA dehydrogenase” refers to an enzyme thatcatalyzes the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA.3-Hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide adeninedinucleotide (NADH)-dependent, with a substrate preference for(S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and are classifiedas E.C. 1.1.1.35 and E.C. 1.1.1.30, respectively. Additionally,3-hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide adeninedinucleotide phosphate (NADPH)-dependent, with a substrate preferencefor (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and areclassified as E.C. 1.1.1.157 and E.C. 1.1.1.36, respectively.3-Hydroxybutyryl-CoA dehydrogenases are available from a number ofsources, for example, C. acetobutylicum (GenBank NOs: NP_(—)349314 (SEQID NO:6), NC_(—)003030 (SEQ ID NO:5)), B. subtilis (GenBank NOs:AAB09614 (SEQ ID NO:135), U29084 (SEQ ID NO:134)), Ralstonia eutropha(GenBank NOs: YP_(—)294481 (SEQ ID NO:137), NC_(—)007347 (SEQ IDNO:136)), and Alcaligenes eutrophus (GenBank NOs: AAA21973 (SEQ IDNO:139), J04987 (SEQ ID NO:138)).

The term “crotonase” refers to an enzyme that catalyzes the conversionof 3-hydroxybutyryl-CoA to crotonyl-CoA and H₂O. Crotonases may have asubstrate preference for (S)-3-hydroxybutyryl-CoA or(R)-3-hydroxybutyryl-CoA and are classified as E.C. 4.2.1.17 and E.C.4.2.1.55, respectively. Crotonases are available from a number ofsources, for example, E. coli (GenBank NOs: NP_(—)415911 (SEQ IDNO:141), NC_(—)000913 (SEQ ID NO:140)), C. acetobutylicum (GenBank NOs:NP_(—)349318 (SEQ ID NO:8), NC_(—)003030 (SEQ ID NO:6)), B. subtilis(GenBank NOs: CAB13705 (SEQ ID NO:143), Z99113 (SEQ ID NO:142)), andAeromonas caviae (GenBank NOs: BAA21816 (SEQ ID NO:145), D88825 (SEQ IDNO:144)).

The term “butyryl-CoA dehydrogenase” refers to an enzyme that catalyzesthe conversion of crotonyl-CoA to butyryl-CoA. Butyryl-CoAdehydrogenases may be either NADH-dependent or NADPH-dependent and areclassified as E.C. 1.3.1.44 and E.C. 1.3.1.38, respectively. Butyryl-CoAdehydrogenases are available from a number of sources, for example, C.acetobutylicum (GenBank NOs: NP_(—)347102 (SEQ ID NO:10), NC_(—)003030(SEQ ID NO:9))), Euglena gracilis (GenBank NOs: Q5EU90 SEQ ID NO:147),AY741582 SEQ ID NO:146)), Streptomyces collinus (GenBank NOs: AAA92890(SEQ ID NO:149), U37135 (SEQ ID NO:148)), and Streptomyces coelico/or(GenBank NOs: CAA22721 (SEQ ID NO:151), AL939127 (SEQ ID NO:150)).

The term “butyraldehyde dehydrogenase” refers to an enzyme thatcatalyzes the conversion of butyryl-CoA to butyraldehyde, using NADH orNADPH as cofactor. Butyraldehyde dehydrogenases with a preference forNADH are known as E.C. 1.2.1.57 and are available from, for example,Clostridium beijerinckii (GenBank NOs: AAD31841 (SEQ ID NO:12), AF157306(SEQ ID NO:11)) and C. acetobutylicum (GenBank NOs: NP_(—)149325 (SEQ IDNO:153), NC_(—)001988 (SEQ ID NO:152)).

The term “butanol dehydrogenase” refers to an enzyme that catalyzes theconversion of butyraldehyde to 1-butanol, using either NADH or NADPH ascofactor. Butanol dehydrogenases are available from, for example, C.acetobutylicum (GenBank NOs: NP_(—)149325 (SEQ ID NO:153), NC_(—)001988SEQ ID NO:152; note: this enzyme possesses both aldehyde and alcoholdehydrogenase activity); NP_(—)349891 (SEQ ID NO:14), NC_(—)003030 (SEQID NO:13); and NP_(—)349892 (SEQ ID NO:16), NC_(—)003030 (SEQ ID NO:15))and E. coli (GenBank NOs: NP_(—)417484 (SEQ ID NO:155), NC_(—)000913(SEQ ID NO:154)).

The term “a facultative anaerobe” refers to a microorganism that cangrow in both aerobic and anaerobic environments.

The term “carbon substrate” or “fermentable carbon substrate” refers toa carbon source capable of being metabolized by host organisms disclosedherein and particularly carbon sources selected from the groupconsisting of monosaccharides, oligosaccharides, polysaccharides, andone-carbon substrates or mixtures thereof.

The term “gene” refers to a nucleic acid fragment that is capable ofbeing expressed as a specific protein, optionally including regulatorysequences preceding (5′ non-coding sequences) and following (3′non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign” or “heterologous gene” refers to a gene notnormally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

As used herein, an “isolated nucleic acid fragment” or “isolated nucleicacid molecule” or “genetic construct” will be used interchangeably andwill mean a polymer of RNA or DNA that is single- or double-stranded,optionally containing synthetic, non-natural or altered nucleotidebases. An isolated nucleic acid fragment in the form of a polymer of DNAmay be comprised of one or more segments of cDNA, genomic DNA orsynthetic DNA.

A nucleic acid fragment is “hybridizable” to another nucleic acidfragment, such as a cDNA, genomic DNA, or RNA molecule, when asingle-stranded form of the nucleic acid fragment can anneal to theother nucleic acid fragment under the appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known and exemplified in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989),particularly Chapter 11 and Table 11.1 therein (entirely incorporatedherein by reference). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. Stringency conditionscan be adjusted to screen for moderately similar fragments (such ashomologous sequences from distantly related organisms), to highlysimilar fragments (such as genes that duplicate functional enzymes fromclosely related organisms). Post-hybridization washes determinestringency conditions. One set of preferred conditions uses a series ofwashes starting with 6×SSC, 0.5% SDS at room temperature for 15 min,then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and thenrepeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A morepreferred set of stringent conditions uses higher temperatures in whichthe washes are identical to those above except for the temperature ofthe final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C.Another preferred set of highly stringent conditions uses two finalwashes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringentconditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washeswith 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see Sambrooket al., supra, 9.50-9.51). For hybridizations with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.7-11.8). In one embodimentthe length for a hybridizable nucleic acid is at least about 10nucleotides. Preferably a minimum length for a hybridizable nucleic acidis at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least about 30nucleotides. Furthermore, the skilled artisan will recognize that thetemperature and wash solution salt concentration may be adjusted asnecessary according to factors such as length of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Altschul, S. F., et al.,J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten ormore contiguous amino acids or thirty or more nucleotides is necessaryin order to putatively identify a polypeptide or nucleic acid sequenceas homologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence. The instant specification teaches the complete amino acidand nucleotide sequence encoding particular fungal proteins. The skilledartisan, having the benefit of the sequences as reported herein, may nowuse all or a substantial portion of the disclosed sequences for purposesknown to those skilled in this art. Accordingly, the instant inventioncomprises the complete sequences as reported in the accompanyingSequence Listing, as well as substantial portions of those sequences asdefined above.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine.

The terms “homology” and “homologous” are used interchangeably herein.They refer to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate gene expression or produce a certain phenotype. These termsalso refer to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotides thatdo not substantially alter the functional properties of the resultingnucleic acid fragment relative to the initial, unmodified fragment. Itis therefore understood, as those skilled in the art will appreciate,that the invention encompasses more than the specific exemplarysequences.

Moreover, the skilled artisan recognizes that homologous nucleic acidsequences encompassed by this invention are also defined by theirability to hybridize, under moderately stringent conditions (e.g.,0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or toany portion of the nucleotide sequences disclosed herein and which arefunctionally equivalent to any of the nucleic acid sequences disclosedherein.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: 1.) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991).

Preferred methods to determine identity are designed to give the bestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the MegAlign™ program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencesis performed using the “Clustal method of alignment” which encompassesseveral varieties of the algorithm including the “Clustal V method ofalignment” corresponding to the alignment method labeled Clustal V(described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in theMegAlign™ program of the LASERGENE bioinformatics computing suite(DNASTAR Inc.). For multiple alignments, the default values correspondto GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters forpairwise alignments and calculation of percent identity of proteinsequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2,GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of thesequences using the Clustal V program, it is possible to obtain a“percent identity” by viewing the “sequence distances” table in the sameprogram. Additionally the “Clustal W method of alignment” is availableand corresponds to the alignment method labeled Clustal W (described byHiggins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al.,Comput. Appl. Biosci. 8:189-191 (1992)) and found in the MegAlign™ v6.1program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.).Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTHPENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5,Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). Afteralignment of the sequences using the Clustal W program, it is possibleto obtain a “percent identity” by viewing the “sequence distances” tablein the same program.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, from otherspecies, wherein such polypeptides have the same or similar function oractivity. Useful examples of percent identities include, but are notlimited to: 24%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95%, or any integer percentage from 24% to 100% may beuseful in describing the present invention, such as 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99%. Suitable nucleic acid fragments not only have the above homologiesbut typically encode a polypeptide having at least 50 amino acids,preferably at least 100 amino acids, more preferably at least 150 aminoacids, still more preferably at least 200 amino acids, and mostpreferably at least 250 amino acids.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.,215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

As used herein the term “coding sequence” refers to a DNA sequence thatcodes for a specific amino acid sequence. “Suitable regulatorysequences” refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing site, effectorbinding site and stem-loop structure.

The term “promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment disclosed herein. Expression may also refer totranslation of mRNA into a polypeptide.

As used herein the term “transformation” refers to the transfer of anucleic acid fragment into a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The terms “plasmid” and “vector” refer to an extra chromosomal elementoften carrying genes which are not part of the central metabolism of thecell, and usually in the form of circular double-stranded DNA molecules.Such elements may be autonomously replicating sequences, genomeintegrating sequences, phage or nucleotide sequences, linear orcircular, of a single- or double-stranded DNA or RNA, derived from anysource, in which a number of nucleotide sequences have been joined orrecombined into a unique construction which is capable of introducing apromoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell. “Transformationvector” refers to a specific vector containing a foreign gene and havingelements in addition to the foreign gene that facilitates transformationof a particular host cell.

As used herein the term “codon degeneracy” refers to the nature in thegenetic code permitting variation of the nucleotide sequence withouteffecting the amino acid sequence of an encoded polypeptide. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L.and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987).

The 1-Butanol Biosynthetic Pathway

Carbohydrate utilizing microorganisms employ the Embden-Meyerhof-Parnas(EMP) pathway, the Entner-Doudoroff pathway and the pentose phosphatecycle as the central, metabolic routes to provide energy and cellularprecursors for growth and maintenance. These pathways have in common theintermediate glyceraldehyde-3-phosphate and, ultimately, pyruvate isformed directly or in combination with the EMP pathway. Subsequently,pyruvate is transformed to acetyl-coenzyme A (acetyl-CoA) via a varietyof means, including reaction with the pyruvate dehydrogenase complex,pyruvate-formate lyase, and pyruvate-ferredoxin oxidoreductase.Acetyl-CoA serves as a key intermediate, for example, in generatingfatty acids, amino acids and secondary metabolites. The combinedreactions of sugar conversion to acetyl-CoA produce energy (e.g.adenosine-5′-triphosphate, ATP) and reducing equivalents (e.g. reducednicotinamide adenine dinucleotide, NADH, and reduced nicotinamideadenine dinucleotide phosphate, NADPH). NADH and NADPH must be recycledto their oxidized forms (NAD⁺ and NADP⁺, respectively). In the presenceof inorganic electron acceptors (e.g. O₂, NO₃ ⁻ and SO₄ ²⁻), thereducing equivalents may be used to augment the energy pool;alternatively, a reduced carbon by-product may be formed. The productionof ethanol and 1-butanol resulting from the fermentation of carbohydrateare examples of the latter. As described by Donaldson, supra, 1-butanolcan be produced from carbohydrate sources by recombinant microorganismscomprising a complete 1-butanol biosynthetic pathway from acetyl-CoA to1-butanol, as shown in FIG. 1.”

This biosynthetic pathway, generally lacking in the microbial communitydue to the absence of genes or the lack of appropriate gene regulation,comprises the following substrate to product conversions:

-   -   a) acetyl-CoA to acetoacetyl-CoA, as catalyzed for example by        acetyl-CoA acetyltransferase;    -   b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, as catalyzed for        example by 3-hydroxybutyryl-CoA dehydrogenase;    -   c) 3-hydroxybutyryl-CoA to crotonyl-CoA, as catalyzed for        example by crotonase;    -   d) crotonyl-CoA to butyryl-CoA, as catalyzed for example by        butyryl-CoA dehydrogenase;    -   e) butyryl-CoA to butyraldehyde, as catalyzed for example by        butyraldehyde dehydrogenase; and    -   f) butyraldehyde to 1-butanol, as catalyzed for example by        butanol dehydrogenase.

The pathway requires no ATP and generates NAD⁺ and/or NADP⁺, thus,balances with the central metabolic routes that generate acetyl-CoA. Theability of natural organisms to produce 1-butanol by fermentation israre and exemplified most prominently by Clostridium beijerinckii andClostridium acetobutylicum. The gene organization and gene regulationfor Clostridium acetobutylicum has been described (L. Girbal and P.Soucaille, Trends in Biotechnology 216:11-16 (1998)). However, many ofthese enzyme activities are associated also with alternate pathways, forexample, hydrocarbon utilization, fatty acid oxidation, andpoly-hydroxyalkanoate metabolism. Thus, in providing a recombinantpathway from acetyl-CoA to 1-butanol, there exist a number of choices tofulfill the individual reaction steps, and the person of skill in theart will be able to utilize publicly available sequences to constructthe relevant pathways. A listing of a representative number of genesknown in the art and useful in the construction of the 1-butanolbiosynthetic pathway are listed below in Table 2 and in Donaldson etal., copending and commonly owned U.S. patent application Ser. No.11/527,995, incorporated herein by reference.

TABLE 2 Sources of 1-Buatnol Pathway Genes Gene GenBank Citationacetyl-CoA NC_000913 Escherichia coli K12, complete genomeacetyltransferase gi|49175990|ref|NC_000913.2|[49175990] NC_001988Clostridium acetobutylicum ATCC 824 plasmid pSOL1, complete sequencegi|15004705|ref|NC_001988.2|[15004705] NC_000964 Bacillus subtilissubsp. subtilis str. 168, complete genomegi|50812173|ref|NC_000964.2|[50812173] NC_001148 Saccharomycescerevisiae chromosome XVI, complete chromosome sequencegi|50593503|ref|NC_001148.3|[50593503] CP000017 Streptococcus pyogenesMGAS5005, complete genome gi|71852596|gb|CP000017.1|[71852596] NC_005773Pseudomonas syringae pv. phaseolicola 1448A, complete genomegi|71733195|ref|NC_005773.3|[71733195] CR931997 Corynebacterium jeikeiumK411 complete genome gi|68262661|emb|CR931997.1|[68262661]3-hydroxybutyryl-CoA NC_003030 Clostridium acetobutylicum ATCC 824,dehydrogenase complete genome gi|15893298|ref|NC_003030.1|[15893298]U29084 Bacillus subtilis (mmgA), (mmgB), (mmgC), and citrate synthaseIII (mmgD) genes, complete cds, and (mmgE) gene, partial cdsgi|881603|gb|U29084.1|BSU29084[881603] NC_007347 Ralstonia eutrophaJMP134 Raeut01_1, whole genome shotgun sequencegi|45517296|ref|NZ_AADY01000001.1|[45517296] J04987 A. eutrophusbeta-ketothiolase (phbA) and acetoacetyl-CoA reductase (phbB) genes,complete cds gi|141953|gb|J04987.1|AFAKTLAACA[141953] NC_004129Pseudomonas fluorescens Pf-5, complete genomegi|70728250|ref|NC_004129.6|[70728250] NC_000913 Escherichia coli K12,complete genome gi|49175990|ref|NC_000913.2|[49175990] NC_004557Clostridium tetani E88, complete genomegi|28209834|ref|NC_004557.1|[28209834] NC_006350 Burkholderiapseudomallei K96243 chromosome 1, complete sequencegi|53717639|ref|NC_006350.1|[53717639] NC_002947 Pseudomonas putidaKT2440, complete genome gi|26986745|ref|NC_002947.3|[26986745] crotonaseNC_000913 Escherichia coli K12, complete genomegi|49175990|ref|NC_000913.2|[49175990] NC_003030 Clostridiumacetobutylicum ATCC 824, complete genomegi|15893298|ref|NC_003030.1|[15893298] Z99113 Bacillus subtilis completegenome (section 10 of 21): from 1807106 to 2014934gi|32468758|emb|Z99113.2|BSUB0010[32468758] D88825 Aeromonas caviae phaCgene for PHA synthase, complete cds gi|2335048|dbj|D88825.1|[2335048]NC_006274 Bacillus cereus ZK, complete genomegi|52140164|ref|NC_006274.1|[52140164] NC_004557 Clostridium tetani E88,complete genome gi|28209834|ref|NC_004557.1|[28209834] butyryl-CoANC_003030 Clostridium acetobutylicum ATCC 824, dehydrogenase completegenome gi|15893298|ref|NC_003030.1|[15893298] AY741582 Euglena gracilistrans-2-enoyl-CoA reductase mRNA, complete cdsgi|58201539|gb|AY741582.1|[58201539] U37135 Streptomyces collinuscrotonyl-CoA reductase (ccr) gene, complete cdsgi|1046370|gb|U37135.1|SCU37135[1046370] AL939127 Streptomycescoelicolor A3(2) complete genome; segment 24/29gi|24429552|emb|AL939127.1|SCO939127[24429552] AP006716 Staphylococcushaemolyticus JCSC1435, complete genomegi|68445725|dbj|AP006716.1|[68445725] NC_006274 Bacillus cereus ZK,complete genome gi|52140164|ref|NC_006274.1|[52140164] NC_004557Clostridium tetani E88, complete genomegi|28209834|ref|NC_004557.1|[28209834] butyraldehyde AF157306Clostridium beijerinckii strain NRRL B593 dehydrogenase hypotheticalprotein, coenzyme A acylating aldehyde dehydrogenase (ald),acetoacetate:butyrate/acetate coenzyme A transferase (ctfA),acetoacetate:butyrate/acetate coenzyme A transferase (ctfB), andacetoacetate decarboxylase (adc) genes, complete cdsgi|47422980|gb|AF157306.2|[47422980] NC_001988 Clostridiumacetobutylicum ATCC 824 plasmid pSOL1, complete sequencegi|15004705|ref|NC_001988.2|[15004705] AY251646 Clostridiumsaccharoperbutylacetonicum sol operon, complete sequencegi|31075382|gb|AY251646.1|[31075382] butanol NC_001988 Clostridiumacetobutylicum ATCC 824 dehydrogenase plasmid pSOL1, complete sequencegi|15004705|ref|NC_001988.2|[15004705] NC_003030 Clostridiumacetobutylicum ATCC 824, complete genomegi|15893298|ref|NC_003030.1|[15893298] NC_000913 Escherichia coli K12,complete genome gi|49175990|ref|NC_000913.2|[49175990] NC_003198Salmonella enterica subsp. enterica serovar Typhi str. CT18, completegenome gi|16758993|ref|NC_003198.1|[16758993] BX571966 Burkholderiapseudomallei strain K96243, chromosome 2, complete sequencegi|52211453|emb|BX571966.1|[52211453 Z99120 Bacillus subtilis completegenome (section 17 of 21): from 3213330 to 3414388gi|32468813|emb|Z99120.2|BSUB0017[32468813 NC_003366 Clostridiumperfringens str. 13, complete genomegi|18308982|ref|NC_003366.1|[18308982 NC_004431 Escherichia coli CFT073,complete genome gi|26245917|ref|NC_004431.1|[26245917

Pathway Steps:

a) Acetyl-CoA to acetoacetyl-CoA, is catalyzed by acetyl-CoAacetyltransferase. The skilled person will appreciate that polypeptideshaving by acetyl-CoA acetyltransferase activity isolated from a varietyof sources will be useful in the present invention independent ofsequence homology. Examples of suitable by acetyl-CoA acetyltransferaseenzymes are available from a number of sources, for example, forexample, Escherichia coli (GenBank Nos: NP_(—)416728 (SEQ ID NO:129),NC_(—)000913 (SEQ ID NO:128); NCBI (National Center for BiotechnologyInformation) amino acid sequence, NCBI nucleotide sequence), Clostridiumacetobutylicum (GenBank Nos: NP_(—)349476.1 (SEQ ID NO:2), NC_(—)003030(SEQ ID NO:1); NP_(—)149242 (SEQ ID NO:4), NC_(—)001988 (SEQ ID NO:3),Bacillus subtilis (GenBank Nos: NP_(—)390297 (SEQ ID NO:131),NC_(—)000964 (SEQ ID NO:130)), and Saccharomyces cerevisiae (GenBankNos: NP_(—)015297 (SEQ ID NO:133), NC_(—)001148 (SEQ ID NO:132)).Preferred by acetyl-CoA acetyltransferase enzymes are those that have atleast 80%-85% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:129, SEQID NO:131, or SEQ ID NO:133, where at least 85%-90% identity is morepreferred and where at least 95% identity based on the Clustal W methodof alignment using the default parameters of GAP PENALTY=10, GAP LENGTHPENALTY=0.1, and Gonnet 250 series of protein weight matrix, is mostpreferred.

b) Acetoacetyl-CoA to 3-hydroxybutyryl-CoA is catalyzed by3-hydroxybutyryl-CoA dehydrogenase. The skilled person will appreciatethat polypeptides having 3-hydroxybutyryl-CoA dehydrogenase activityisolated from a variety of sources will be useful in the presentinvention independent of sequence homology. Example of suitable3-hydroxybutyryl-CoA dehydrogenase enzymes are available from a numberof sources, for example, C. acetobutylicum (GenBank NOs: NP_(—)349314(SEQ ID NO:6), NC_(—)003030 (SEQ ID NO:5)), B. subtilis (GenBank NOs:AAB09614 (SEQ ID NO:135), U29084 (SEQ ID NO:134)), Ralstonia eutropha(GenBank NOs: YP_(—)294481 (SEQ ID NO:137), NC_(—)007347 (SEQ IDNO:136)), and Alcaligenes eutrophus (GenBank NOs: AAA21973 (SEQ IDNO:139), J04987 (SEQ ID NO:138)). Preferred 3-hydroxybutyryl-CoAdehydrogenase enzymes are those that have at least 80%-85% identity toSEQ ID NO:6, SEQ ID NO:135, SEQ ID NO:137, or SEQ ID NO:139 where atleast 85%-90% identity is more preferred and where at least 95% identitybased on the Clustal W method of alignment using the default parametersof GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series ofprotein weight matrix, is most preferred.

c) 3-hydroxybutyryl-CoA to crotonyl-CoA is catalyzed by crotonase. Theskilled person will appreciate that polypeptides having crotonaseactivity isolated from a variety of sources will be useful in thepresent invention independent of sequence homology. Examples of suitablecrotonase enzymes are available from a number of sources, for example,E. coli (GenBank NOs: NP_(—)415911 (SEQ ID NO:141), NC_(—)000913 (SEQ IDNO:140)), C. acetobutylicum (GenBank NOs: NP_(—)349318 (SEQ ID NO:8),NC_(—)003030 (SEQ ID NO:6)), B. subtilis (GenBank NOs: CAB13705 (SEQ IDNO:143), Z99113 (SEQ ID NO:142)), and Aeromonas caviae (GenBank NOs:BAA21816 (SEQ ID NO:145), D88825 (SEQ ID NO:144)). Preferred crotonaseenzymes are those that have at least 80%-85% identity to SEQ ID NO:8,SEQ ID NO:141, SEQ ID NO:143, and SEQ ID NO:145 where at least 85%-90%identity is more preferred and where at least 95% identity based on theClustal W method of alignment using the default parameters of GAPPENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of proteinweight matrix, is most preferred.

d) Crotonyl-CoA to butyryl-CoA, is catalyzed by butyryl-CoAdehydrogenase. The skilled person will appreciate that polypeptideshaving butyryl-CoA dehydrogenase activity isolated from a variety ofsources will be useful in the present invention independent of sequencehomology. Examples of suitable butyryl-CoA dehydrogenase enzymes areavailable from a number of sources, for example, C. acetobutylicum(GenBank NOs: NP_(—)347102 (SEQ ID NO:10), NC_(—)003030 (SEQ ID NO:9))),Euglena gracilis (GenBank NOs: Q5EU90 SEQ ID NO:147), AY741582 SEQ IDNO:146)), Streptomyces collinus (GenBank NOs: AAA92890 (SEQ ID NO:149),U37135 (SEQ ID NO:148)), and Streptomyces coelicolor (GenBank NOs:CAA22721 (SEQ ID NO:151), AL939127 (SEQ ID NO:150)). Preferredbutyryl-CoA dehydrogenase enzymes are those that have at least 80%-85%identity to SEQ ID NO:10, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,and SEQ ID NO:187 where at least 85%-90% identity is more preferred andwhere at least 95% identity based on the Clustal W method of alignmentusing the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1,and Gonnet 250 series of protein weight matrix, is most preferred.

e) Butyryl-CoA to butyraldehyde, is catalyzed by butyraldehydedehydrogenase. The skilled person will appreciate that polypeptideshaving butyraldehyde dehydrogenase activity isolated from a variety ofsources will be useful in the present invention independent of sequencehomology. Examples of suitable butyraldehyde dehydrogenase enzymes areavailable from a number of sources, for example, Clostridiumbeijerinckii (GenBank NOs: AAD31841 (SEQ ID NO:12), AF157306 (SEQ IDNO:11)) and C. acetobutylicum (GenBank NOs: NP_(—)149325 (SEQ IDNO:153), NC_(—)001988 (SEQ ID NO:152)). Preferred butyraldehydedehydrogenase enzymes are those that have at least 80%-85% identity toSEQ ID NO:12, SEQ ID NO:153, and SEQ ID NO:189 where at least 85%-90%identity is more preferred and where at least 95% identity based on theClustal W method of alignment using the default parameters of GAPPENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of proteinweight matrix, is most preferred.

f) Butyraldehyde to 1-butanol, is catalyzed by butanol dehydrogenase.The skilled person will appreciate that polypeptides having butanoldehydrogenase activity isolated from a variety of sources will be usefulin the present invention independent of sequence homology. Some exampleof suitable butanol dehydrogenase enzymes are available from a number ofsources, for example, C. acetobutylicum (GenBank NOs: NP_(—)149325 (SEQID NO:153), NC_(—)001988 SEQ ID NO:152; note: this enzyme possesses bothaldehyde and alcohol dehydrogenase activity); NP_(—)349891 (SEQ IDNO:14), NC_(—)003030 (SEQ ID NO:13); and NP_(—)349892 (SEQ ID NO:16),NC_(—)003030 (SEQ ID NO:15)) and E. coli (GenBank NOs: NP_(—)417484 (SEQID NO:155), NC_(—)000913 (SEQ ID NO:154)). Preferred butanoldehydrogenase enzymes are those that have at least 80%-85% identity toSEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:153, SEQ ID NO:155, and SEQ IDNO:157 where at least 85%-90% identity is more preferred and where atleast 95% identity based on the Clustal W method of alignment using thedefault parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet250 series of protein weight matrix, is most preferred.

Microbial Hosts for 1-Butanol Production

Microbial hosts for 1-butanol production may be selected from bacteria,cyanobacteria, filamentous fungi and yeasts. The microbial host used for1-butanol production is preferably tolerant to 1-butanol so that theyield is not limited by butanol toxicity. Microbes that aremetabolically active at high titer levels of 1-butanol are not wellknown in the art. Although 1-butanol-tolerant mutants have been isolatedfrom solventogenic Clostridia, little information is availableconcerning the 1-butanol tolerance of other potentially useful bacterialstrains. Most of the studies on the comparison of alcohol tolerance inbacteria suggest that 1-butanol is more toxic than ethanol (de Cavalhoet al., Microsc. Res. Tech. 64:215-22 (2004) and Kabelitz et al., FEMSMicrobiol. Lett. 220:223-227 (2003)). Tomas et al. (J. Bacteriol.186:2006-2018 (2004)) report that the yield of 1-butanol duringfermentation in Clostridium acetobutylicum may be limited by toxicity.The primary effect of 1-butanol on Clostridium acetobutylicum isdisruption of membrane functions (Hermann et al., Appl. Environ.Microbiol. 50:1238-1243 (1985)).

The microbial hosts selected for the production of 1-butanol arepreferably tolerant to 1-butanol and are able to convert carbohydratesto 1-butanol. The criteria for selection of suitable microbial hostsinclude the following: intrinsic tolerance to 1-butanol, high rate ofglucose utilization, availability of genetic tools for genemanipulation, and the ability to generate stable chromosomalalterations.

Suitable host strains with a tolerance for 1-butanol may be identifiedby screening based on the intrinsic tolerance of the strain. Theintrinsic tolerance of microbes to 1-butanol may be measured bydetermining the concentration of 1-butanol that is responsible for 50%inhibition of the growth rate (IC50) when grown in a minimal medium. TheIC50 values may be determined using methods known in the art. Forexample, the microbes of interest may be grown in the presence ofvarious amounts of 1-butanol and the growth rate monitored by measuringthe optical density at 600 nanometers. The doubling time may becalculated from the logarithmic part of the growth curve and used as ameasure of the growth rate. The concentration of 1-butanol that produces50% inhibition of growth may be determined from a graph of the percentinhibition of growth versus the 1-butanol concentration. Preferably, thehost strain should have an IC50 for 1-butanol of greater than about 0.5%weight/volume.

The microbial host for 1-butanol production should also utilize glucoseat a high rate. Most microbes are capable of utilizing carbohydrates.However, certain environmental microbes cannot utilize carbohydrates tohigh efficiency, and therefore would not be suitable hosts.

The ability to genetically modify the host is essential for theproduction of any recombinant microorganism. The mode of gene transfertechnology may be by electroporation, conjugation, transduction ornatural transformation. A broad range of host conjugative plasmids anddrug resistant markers are available. The cloning vectors are tailoredto the host organisms based on the nature of antibiotic resistancemarkers that can function in that host.

The microbial host also has to be manipulated in order to inactivatecompeting pathways for carbon flow by deleting various genes. Thisrequires the availability of either transposons to direct inactivationor chromosomal integration vectors. Additionally, the production hostshould be amenable to chemical mutagenesis so that mutations to improveintrinsic 1-butanol tolerance may be obtained.

Based on the criteria described above, suitable microbial hosts for theproduction of 1-butanol include, but are not limited to, members of thegenera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus,Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes,Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. Preferredhosts include: Escherichia coli, Alcaligenes eutrophus, Bacilluslicheniformis, Paenibacillus macerans, Rhodococcus erythropolis,Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium,Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis andSaccharomyces cerevisiae.

Construction of Production Host

Recombinant organisms containing the necessary genes that will encodethe enzymatic pathway for the conversion of a fermentable carbonsubstrate to 1-butanol may be constructed using techniques well known inthe art. Genes encoding the enzymes of the 1-butanol biosyntheticpathway, i.e., acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoAdehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehydedehydrogenase, and butanol dehydrogenase, may be isolated from varioussources, as described above.

Methods of obtaining desired genes from a bacterial genome are commonand well known in the art of molecular biology. For example, if thesequence of the gene is known, suitable genomic libraries may be createdby restriction endonuclease digestion and may be screened with probescomplementary to the desired gene sequence. Once the sequence isisolated, the DNA may be amplified using standard primer-directedamplification methods such as polymerase chain reaction (Mullis, U.S.Pat. No. 4,683,202) to obtain amounts of DNA suitable for transformationusing appropriate vectors. Tools for codon optimization for expressionin a heterologous host are readily available. Some tools for codonoptimization are available based on the GC content of the host organism.The GC content of some exemplary microbial hosts is given Table 3.

TABLE 3 GC Content of Microbial Hosts Strain % GC B. licheniformis 46 B.subtilis 42 C. acetobutylicum 37 E. coli 50 P. putida 61 A. eutrophus 61Paenibacillus macerans 51 Rhodococcus erythropolis 62 Brevibacillus 50Paenibacillus polymyxa 50

Once the relevant pathway genes are identified and isolated they may betransformed into suitable expression hosts by means well known in theart. Vectors or cassettes useful for the transformation of a variety ofhost cells are common and commercially available from companies such asEPICENTRE® (Madison, Wis.), Invitrogen Corp. (Carlsbad, Calif.),Stratagene (La Jolla, Calif.), and New England Biolabs, Inc. (Beverly,Mass.). Typically, the vector or cassette contains sequences directingtranscription and translation of the relevant gene, a selectable marker,and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene whichharbors transcriptional initiation controls and a region 3′ of the DNAfragment which controls transcriptional termination. Both controlregions may be derived from genes homologous to the transformed hostcell, although it is to be understood that such control regions may alsobe derived from genes that are not native to the specific species chosenas a production host.

Initiation control regions or promoters, which are useful to driveexpression of the relevant pathway coding regions in the desired hostcell are numerous and familiar to those skilled in the art. Virtuallyany promoter capable of driving these genetic elements is suitable forthe present invention including, but not limited to, CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, CUP1,FBA, GPD, and GPM (useful for expression in Saccharomyces); AOX1 (usefulfor expression in Pichia); and lac, ara, tet, trp, IP_(L), IP_(R), T7,tac, and trc (useful for expression in Escherichia coli, Alcaligenes,and Pseudomonas); the amy, apr, npr promoters and various phagepromoters useful for expression in Bacillus subtilis, Bacilluslicheniformis, and Paenibacillus macerans; nisA (useful for expressionGram-positive bacteria, Eichenbaum et al. Appl. Environ. Microbiol.64(8):2763-2769 (1998)); and the synthetic P11 promoter (useful forexpression in Lactobacillus plantarum, Rud et al., Microbiology152:1011-1019 (2006)).

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary, however, it is most preferred if included.

Certain vectors are capable of replicating in a broad range of hostbacteria and can be transferred by conjugation. The complete andannotated sequence of pRK404 and three related vectors-pRK437, pRK442,and pRK442(H) are available. These derivatives have proven to bevaluable tools for genetic manipulation in Gram-negative bacteria (Scottet al., Plasmid 50(1):74-79 (2003)). Several plasmid derivatives ofbroad-host-range Inc P4 plasmid RSF1010 are also available withpromoters that can function in a range of Gram-negative bacteria.Plasmid pAYC36 and pAYC37, have active promoters along with multiplecloning sites to allow for the heterologous gene expression inGram-negative bacteria.

Chromosomal gene replacement tools are also widely available. Forexample, a thermosensitive variant of the broad-host-range repliconpWV101 has been modified to construct a plasmid pVE6002 which can beused to create gene replacement in a range of Gram-positive bacteria(Maguin et al., J. Bacteriol. 174(17):5633-5638 (1992)). Additionally,in vitro transposomes are available to create random mutations in avariety of genomes from commercial sources such as EPICENTRE®.

The expression of the 1-butanol biosynthetic pathway in variouspreferred microbial hosts is described in more detail below.

Expression of the 1-Butanol Biosynthetic Pathway in E. Coli

Vectors or cassettes useful for the transformation of E. coli are commonand commercially available from the companies listed above. For example,the genes of the 1-butanol biosynthetic pathway may be isolated fromvarious strains of Clostridium, cloned into a modified pUC19 vector andtransformed into E. coli NM522, as described in Example 15. Theexpression of the 1-butanol biosynthetic pathway in several otherstrains of E. coli is described in Example 17.

Expression of the 1-Butanol Biosynthetic Pathway in RhodococcusErythrodolis

A series of E. coli-Rhodococcus shuttle vectors are available forexpression in R. erythropolis, including, but not limited to pRhBR17 andpDA71 (Kostichka et al., Appl. Microbiol. Biotechno/62:61-68 (2003)).Additionally, a series of promoters are available for heterologous geneexpression in R. erythropolis (see for example Nakashima et al., Appl.Envir. Microbiol. 70:5557-5568 (2004), and Tao et al., Appl. Microbiol.Biotechnol. 2005, DOI 10.1007/s00253-005-0064). Targeted gene disruptionof chromosomal genes in R. erythropolis may be created using the methoddescribed by Tao et al., supra, and Brans et al. (Appl. Envir.Microbiol. 66: 2029-2036 (2000)).

The heterologous genes required for the production of 1-butanol, asdescribed above, may be cloned initially in pDA71 or pRhBR71 andtransformed into E. coli. The vectors may then be transformed into R.erythropolis by electroporation, as described by Kostichka et al.,supra. The recombinants may be grown in synthetic medium containingglucose and the production of 1-butanol can be followed using methodsknown in the art.

Expression of the 1-Butanol Biosynthetic Pathway in Bacillus subtilis

Methods for gene expression and creation of mutations in B. Subtilis arealso well known in the art. For example, the genes of the 1-butanolbiosynthetic pathway may be isolated from various strains ofClostridium, cloned into a modified pUC19 vector and transformed intoBacillus subtilis BE1010, as described in Example 16. Additionally, thesix genes of the 1-biosynthetic pathway can be split into two operonsfor expression, as described in Example 18. The first three genes of thepathway (thI, hbd, and crt) were integrated into the chromosome ofBacillus subtilis BE1010 (Payne and Jackson, J. Bacteriol. 173:2278-2282(1991)). The last three genes (EgTER, aid, and bdhB) were cloned intoexpression plasmids and transformed into the Bacillus strain carryingthe integrated 1-butanol genes

Expression of the 1-Butanol Biosynthetic Pathway in Bacilluslicheniformis

Most of the plasmids and shuttle vectors that replicate in B. subtilismay be used to transform B. licheniformis by either protoplasttransformation or electroporation. For example, the genes required forthe production of 1-butanol may be cloned in plasmids pBE20 or pBE60derivatives (Nagarajan et al., Gene 114:121-126 (1992)). Methods totransform B. licheniformis are known in the art (for example see Fleminget al. Appl. Environ. Microbiol., 61(11):3775-3780 (1995)). The plasmidsconstructed for expression in B. subtilis may also be transformed intoB. licheniformis to produce a recombinant microbial host that produces1-butanol.

Expression of the 1-Butanol Biosynthetic Pathway in Paenibacillusmacerans

Plasmids may be constructed as described above for expression in B.subtilis and used to transform Paenibacillus macerans by protoplasttransformation to produce a recombinant microbial host that produces1-butanol.

Expression of the 1-Butanol Biosynthetic Pathway in Alcaligenes(Ralstonia) eutrophus

Methods for gene expression and creation of mutations in Ralstoniaeutrophus are known in the art (see for example Taghavi et al., Appl.Environ. Microbiol., 60(10):3585-3591 (1994)). The genes for the1-butanol biosynthetic pathway may be cloned in any of the broad hostrange vectors described above, and electroporated to generaterecombinants that produce 1-butanol. The polyhydroxy butyrate pathway inRalstonia has been described in detail and a variety of genetictechniques to modify the Ralstonia eutrophus genome is known, and thosetools can be applied for engineering the 1-butanol biosynthetic pathway.

Expression of the 1-Butanol Biosynthetic Pathway in Pseudomonas Putida

Methods for gene expression in Pseudomonas putida are known in the art(see for example Ben-Bassat et al., U.S. Pat. No. 6,586,229, which isincorporated herein by reference). For example, the 1-butanol pathwaygenes may be inserted into pPCU18 and this ligated DNA may beelectroporated into electrocompetent Pseudomonas putida DOT-T1 C5aAR1cells to generate recombinants that produce 1-butanol.

Expression of the 1-Butanol Biosynthetic Pathway in Saccharomycescerevisiae

Methods for gene expression in Saccharomyces cerevisiae are known in theart (see for example Methods in Enzymology, Volume 194, Guide to YeastGenetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrieand Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.).Expression of genes in yeast typically requires a promoter, followed bythe gene of interest, and a transcriptional terminator. A number ofyeast promoters can be used in constructing expression cassettes forgenes encoding the 1-butanol biosynthetic pathway, including, but notlimited to constitutive promoters FBA, GPD, and GPM, and the induciblepromoters GAL1, GAL10, and CUP1. Suitable transcriptional terminatorsinclude, but are not limited to FBAt, GPDt, GPMt, ERG10t, and GAL1t.Suitable promoters, transcriptional terminators, and the genes of the1-butanol biosynthetic pathway may be cloned into yeast 2 micron (2μ)plasmids, as described in Example 21.

Expression of the 1-Butanol Biosynthetic Pathway in Lactobacillusplantarum

The Lactobacillus genus belongs to the Lactobacillales family and manyplasmids and vectors used in the transformation of Bacillus subtilis andStreptococcus may be used for lactobacillus. Non-limiting examples ofsuitable vectors include pAMβ1 and derivatives thereof (Renault et al.,Gene 183:175-182 (1996); and O'Sullivan et al., Gene 137:227-231(1993)); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al. Appl.Environ. Microbiol. 62:1481-1486 (1996)); pMGI, a conjugative plasmid(Tanimoto et al., J. Bacteriol. 184:5800-5804 (2002)); pNZ9520(Kleerebezem et al., Appl. Environ. Microbiol. 63:4581-4584 (1997));pAM401 (Fujimoto et al., Appl. Environ. Microbiol. 67:1262-1267 (2001));and pAT392 (Arthur et al., Antimicrob. Agents Chemother. 38:1899-1903(1994)). Several plasmids from Lactobacillus plantarum have also beenreported (e.g., van Kranenburg R, Golic N, Bongers R, Leer R J, de Vos WM, Siezen R J, Kleerebezem M. Appl. Environ. Microbiol. 2005 March;71(3): 1223-1230). For example, expression of the 1-butanol biosyntheticpathway in Lactobacillus plantarum is described in Example 22.

Expression of the 1-Butanol Biosynthetic Pathway in Enterococcusfaecium, Enterococcus gallinarium, and Enterococcus faecalis

The Enterococcus genus belongs to the Lactobacillales family and manyplasmids and vectors used in the transformation of Lactobacillus,Bacillus subtilis, and Streptococcus may be used for Enterococcus.Non-limiting examples of suitable vectors include pAMβ1 and derivativesthereof (Renault et al., Gene 183:175-182 (1996); and O'Sullivan et al.,Gene 137:227-231 (1993)); pMBB1 and pHW800, a derivative of pMBB1(Wyckoff et al. Appl. Environ. Microbiol. 62:1481-1486 (1996)); pMGI, aconjugative plasmid (Tanimoto et al., J. Bacteriol. 184:5800-5804(2002)); pNZ9520 (Kleerebezem et al., Appl. Environ. Microbiol.63:4581-4584 (1997)); pAM401 (Fujimoto et al., Appl. Environ. Microbiol.67:1262-1267 (2001)); and pAT392 (Arthur et al., Antimicrob. AgentsChemother. 38:1899-1903 (1994)). Expression vectors for E. faecalisusing the nisA gene from Lactococcus may also be used (Eichenbaum etal., Appl. Environ. Microbiol. 64:2763-2769 (1998). Additionally,vectors for gene replacement in the E. faecium chromosome may be used(Nallaapareddy et al., Appl. Environ. Microbiol. 72:334-345 (2006)). Forexample, expression of the 1-butanol biosynthetic pathway inEnterococcus faecalis is described in Example 23.

Fermentation Media

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include but are not limited tomonosaccharides such as glucose and fructose, oligosaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, or methanol for which metabolicconversion into key biochemical intermediates has been demonstrated. Inaddition to one and two carbon substrates methylotrophic organisms arealso known to utilize a number of other carbon containing compounds suchas methylamine, glucosamine and a variety of amino acids for metabolicactivity. For example, methylotrophic yeast are known to utilize thecarbon from methylamine to form trehalose or glycerol (Bellion et al.,Microb. Growth C ₁ Compd., [Int. Symp.], 7th (1993), 415-32. Editor(s):Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK).Similarly, various species of Candida will metabolize alanine or oleicacid (Sutter et al., Arch. Microbiol. 153:485-489 (1990)). Hence it iscontemplated that the source of carbon utilized in the present inventionmay encompass a wide variety of carbon containing substrates and willonly be limited by the choice of organism.

Although it is contemplated that all of the above mentioned carbonsubstrates and mixtures thereof are suitable in the present invention,preferred carbon substrates are glucose, fructose, and sucrose. Sucrosemay be derived from renewable sugar sources such as sugar cane, sugarbeets, cassava, sweet sorghum, and mixtures thereof. Glucose anddextrose may be derived from renewable grain sources throughsaccharification of starch based feedstocks including grains such ascorn, wheat, rye, barley, oats, and mixtures thereof. In addition,fermentable sugars may be derived from renewable cellulosic orlignocellulosic biomass through processes of pretreatment andsaccharification, as described, for example, in co-owned and co-pendingU.S. Patent Application Publication No. 2007/0031918A1, which is hereinincorporated by reference. Biomass refers to any cellulosic orlignocellulosic material and includes materials comprising cellulose,and optionally further comprising hemicellulose, lignin, starch,oligosaccharides and/or monosaccharides. Biomass may also compriseadditional components, such as protein and/or lipid. Biomass may bederived from a single source, or biomass can comprise a mixture derivedfrom more than one source; for example, biomass may comprise a mixtureof corn cobs and corn stover, or a mixture of grass and leaves. Biomassincludes, but is not limited to, bioenergy crops, agricultural residues,municipal solid waste, industrial solid waste, sludge from papermanufacture, yard waste, wood and forestry waste. Examples of biomassinclude, but are not limited to, corn grain, corn cobs, crop residuessuch as corn husks, corn stover, grasses, wheat, wheat straw, barley,barley straw, hay, rice straw, switchgrass, waste paper, sugar canebagasse, sorghum, soy, components obtained from milling of grains,trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes,vegetables, fruits, flowers, animal manure, and mixtures thereof.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the enzymatic pathway necessary for1-butanol production.

Culture Conditions with Temperature Lowering

In the present method, the recombinant microbial production host whichproduces 1-butanol is seeded into a fermentation medium comprising afermentable carbon substrate to create a fermentation culture. Theproduction host is grown in the fermentation culture at a firsttemperature for a first period of time. The first temperature istypically from about 25° C. to about 40° C.

Suitable fermentation media in the present invention include commoncommercially prepared media such as Luria Bertani (LB) broth, SabouraudDextrose (SD) broth or Yeast Medium (YM) broth. Other defined orsynthetic growth media may also be used, and the appropriate medium forgrowth of the particular microorganism will be known by one skilled inthe art of microbiology or fermentation science. The use of agents knownto modulate catabolite repression directly or indirectly, e.g., cyclicadenosine 2′:3′-monophosphate, may also be incorporated into thefermentation medium.

Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0,where pH 6.0 to pH 8.0 is preferred as the initial condition.

Fermentations may be performed under aerobic or anaerobic conditions,where anaerobic or microaerobic conditions are preferred.

The first period of time to grow the production host at the firsttemperature may be determined in a variety of ways. For example, duringthis period of growth a metabolic parameter of the fermentation culturemay be monitored. The metabolic parameter that is monitored may be anyparameter known in the art, including, but not limited to the opticaldensity, pH, respiratory quotient, fermentable carbon substrateutilization, CO₂ production, and 1-butanol production. During thisperiod of growth, additional fermentable carbon substrate may be added,the pH may be adjusted, oxygen may be added for aerobic cells, or otherculture parameters may be adjusted to support the metabolic activity ofthe culture. Though nutrients and culture conditions are supportive ofgrowth, after a period of time the metabolic activity of thefermentation culture decreases as determined by the monitored parameterdescribed above. For example, a decrease in metabolic activity may beindicated by a decrease in one or more of the following parameters: rateof optical density change, rate of pH change, rate of change inrespiratory quotient (if the host cells are aerobic), rate offermentable carbon substrate utilization, rate of 1-butanol production,rate of change in CO₂ production, or rate of another metabolicparameter. The decrease in metabolic activity is related to thesensitivity of the host cells to the production of 1-butanol and/or thepresence of 1-butanol in the culture. When decreased metabolic activityis detected, the temperature of the fermentation culture is lowered toreduce the sensitivity of the host cells to 1-butanol and thereby allowfurther production of 1-butanol. In one embodiment, the lowering of thetemperature coincides with a change in the metabolic parameter that ismonitored.

In one embodiment, the change in metabolic activity is a decrease in therate of 1-butanol production. 1-Butanol production may be monitored byanalyzing the amount of 1-butanol present in the fermentation culturemedium as a function of time using methods well known in the art, suchas using high performance liquid chromatography (HPLC) or gaschromatography (GC), which are described in the Examples herein. GC ispreferred due to the short assay time.

Alternatively, the lowering of the temperature of the fermentationculture may occur at a predetermined time. The first period of time maybe predetermined by establishing a correlation between a metabolicparameter of the fermentation culture and time in a series of testfermentations runs. A correlation between a metabolic parameter, asdescribed above, and time of culture growth may be established for any1-butanol producing host by one skilled in the art. The specificcorrelation may vary depending on conditions used including, but notlimited to, carbon substrate, fermentation conditions, and the specificrecombinant 1-butanol producing microbial production host. Thecorrelation is most suitably made between 1-butanol production orspecific glucose consumption rate and time of culture growth. Once thepredetermined time has been established from the correlation, thetemperature of the fermentation culture in subsequent fermentation runsis lowered at the predetermined time. For example, if it is determinedby monitoring a metabolic parameter in the test fermentation runs thatthe rate of production of 1-butanol decreases after 12 hours, thetemperature in subsequent fermentations runs is lowered after 12 hourswithout the need to monitor 1-butanol production in the subsequent runs.

After the first period of time, the temperature of the fermentationculture is lowered to a second temperature. Typically, the secondtemperature is about 3° C. to about 25° C. lower than the firsttemperature. Reduction in temperature to enhance tolerance of the hostcells to 1-butanol is balanced with maintaining the temperature at alevel where the cells continue to be metabolically active for 1-butanolproduction. For example, a fermentation culture that has been grown atabout 35° C. may be reduced in temperature to about 28° C.; or a culturegrown at about 30° C. may be reduced in temperature to about 25° C. Thechange in temperature may be done gradually over time or may be made asa step change. The production host is incubated at the secondtemperature for a second period of time, so that 1-butanol productioncontinues. The second period of time may be determined in the samemanner as the first period of time described above, e.g., by monitoringa metabolic parameter or by using a predetermined time.

Additionally, the temperature lowering and incubation steps may berepeated one or more times to more finely balance metabolic activity for1-butanol production and 1-butanol sensitivity. For example, a culturethat has been grown at about 35° C. may be reduced in temperature toabout 32° C., followed by an incubation period. During this period ametabolic parameter of the fermentation culture may be monitored asdescribed above, or a predetermined time may be used. It is particularlysuitable to monitor the production of 1-butanol during this incubationperiod. When monitoring indicates a decrease in metabolic activity or ata predetermined time, the temperature may be reduced a second time. Forexample, the temperature may be reduced from about 32° C. to about 28°C. The temperature lowering and incubation steps may be repeated a thirdtime where the temperature is reduced, for example, to about 20° C. Theproduction host is incubated at the lowered temperature so that1-butanol production continues. The steps may be repeated further asnecessary to obtain the desired 1-butanol titer.

Industrial Batch and Continuous Fermentations

The present process employs a batch method of fermentation. A classicalbatch fermentation is a closed system where the composition of themedium is set at the beginning of the fermentation and not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the medium is inoculated with the desired organismor organisms, and fermentation is permitted to occur without addinganything to the system. Typically, however, a “batch” fermentation isbatch with respect to the addition of carbon source and attempts areoften made at controlling factors such as pH and oxygen concentration.In batch systems the metabolite and biomass compositions of the systemchange constantly up to the time the fermentation is stopped. Withinbatch cultures cells moderate through a static lag phase to a highgrowth log phase and finally to a stationary phase where growth rate isdiminished or halted. If untreated, cells in the stationary phase willeventually die. Cells in log phase generally are responsible for thebulk of production of end product or intermediate.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-Batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the media. Measurement of the actualsubstrate concentration in Fed-Batch systems is difficult and istherefore estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO₂. Batch and Fed-Batch fermentations are common and well knownin the art and examples may be found in Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, Second Edition(1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, MukundV., Appl. Biochem. Biotechnol., 36:227, (1992), herein incorporated byreference.

Although the present invention is performed in batch mode it iscontemplated that the method would be adaptable to continuousfermentation methods. Continuous fermentation is an open system where adefined fermentation medium is added continuously to a bioreactor and anequal amount of conditioned media is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or end product concentration.For example, one method will maintain a limiting nutrient such as thecarbon source or nitrogen level at a fixed rate and allow all otherparameters to moderate. In other systems a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Continuous systems striveto maintain steady state growth conditions and thus the cell loss due tothe medium being drawn off must be balanced against the cell growth ratein the fermentation. Methods of modulating nutrients and growth factorsfor continuous fermentation processes as well as techniques formaximizing the rate of product formation are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock,supra.

It is contemplated that the present invention may be practiced usingeither batch, fed-batch or continuous processes and that any known modeof fermentation would be suitable. Additionally, it is contemplated thatcells may be immobilized on a substrate as whole cell catalysts andsubjected to fermentation conditions for 1-butanol production.

Methods for 1-Butanol Isolation from the Fermentation Medium

The bioproduced 1-butanol may be isolated from the fermentation mediumusing methods known in the art. For example, solids may be removed fromthe fermentation medium by centrifugation, filtration, decantation, orthe like. Then, the 1-butanol may be isolated from the fermentationmedium, which has been treated to remove solids as described above,using methods such as distillation, liquid-liquid extraction, ormembrane-based separation. Because 1-butanol forms a low boiling point,azeotropic mixture with water, distillation can only be used to separatethe mixture up to its azeotropic composition. Distillation may be usedin combination with another separation method to obtain separationaround the azeotrope. Methods that may be used in combination withdistillation to isolate and purify 1-butanol include, but are notlimited to, decantation, liquid-liquid extraction, adsorption, andmembrane-based techniques. Additionally, 1-butanol may be isolated usingazeotropic distillation using an entrainer (see for example Doherty andMalone, Conceptual Design of Distillation Systems, McGraw Hill, NewYork, 2001).

The 1-butanol-water mixture forms a heterogeneous azeotrope so thatdistillation may be used in combination with decantation to isolate andpurify the 1-butanol. In this method, the 1-butanol containingfermentation broth is distilled to near the azeotropic composition.Then, the azeotropic mixture is condensed, and the 1-butanol isseparated from the fermentation medium by decantation. The decantedaqueous phase may be returned to the first distillation column asreflux. The 1-butanol-rich decanted organic phase may be furtherpurified by distillation in a second distillation column.

The 1-butanol may also be isolated from the fermentation medium usingliquid-liquid extraction in combination with distillation. In thismethod, the 1-butanol is extracted from the fermentation broth usingliquid-liquid extraction with a suitable solvent. The1-butanol-containing organic phase is then distilled to separate the1-butanol from the solvent.

Distillation in combination with adsorption may also be used to isolate1-butanol from the fermentation medium. In this method, the fermentationbroth containing the 1-butanol is distilled to near the azeotropiccomposition and then the remaining water is removed by use of anadsorbent, such as molecular sieves (Aden et al. Lignocellulosic Biomassto Ethanol Process Design and Economics Utilizing Co-Current Dilute AcidPrehydrolysis and Enzymatic Hydrolysis for Corn Stover, ReportNREL/TP-510-32438, National Renewable Energy Laboratory, June 2002).

Additionally, distillation in combination with pervaporation may be usedto isolate and purify the 1-butanol from the fermentation medium. Inthis method, the fermentation broth containing the 1-butanol isdistilled to near the azeotropic composition, and then the remainingwater is removed by pervaporation through a hydrophilic membrane (Guo etal., J. Membr. Sci. 245, 199-210 (2004)).

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989)(Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents, restriction enzymes andmaterials used for the growth and maintenance of bacterial cells wereobtained from Aldrich Chemicals (Milwaukee, Wis.), BD Diagnostic Systems(Sparks, Md.), Life Technologies (Rockville, Md.), or Sigma ChemicalCompany (St. Louis, Mo.) unless otherwise specified. Microbial sampleswere obtained from The American Type Culture Collection (ATCC; Manassas,Va.) unless otherwise noted.

The oligonucleotide primers used for cloning in the following Examplesare given in Table 4. The primers used to sequence or screen the clonedgenes are given in Table 5. All the oligonucleotide primers weresynthesized by Sigma-Genosys (Woodlands, Tex.).

TABLE 4  Oligonucleotide Cloning Primers Primer SEQ ID Gene NameSequence NO: Description crt N3 CACCATGGAACTAAACA 17 crt ATGTCATCCTTGforward crt N4 CCTCCTATCTATTTTTGA 18 crt AGCCTTC reverse hbd N5CACCATGAAAAAGGTAT 19 hbd GTGTTATAGGT forward hbd N6 CATTTGATAATGGGGAT 20hbd TCTTGT reverse thlA N7 CACCATGAAAGAAGTTG 21 thlA TAATAGCTAGTGCforward thlA N8 CTAGCACTTTTCTAGCA 22 thlA ATATTGCTG reverse bdhA N9CACCATGCTAAGTTTTG 23 bdhA ATTATTCAATAC forward bdhA N10TTAATAAGATTTTTTAAA 24 bdhA TATCTCA reverse bdhB N11 CACCATGGTTGATTTCG 25bdhB AATATTCAATACC forward bdhB N12 TTACACAGATTTTTTGAA 26 bdhB TATTTGTreverse thlB N15 CACCATGAGAGATGTAG 27 thlB TAATAGTAAGTGCTG forward thlBN16 CCGCAATTGTATCCATA 28 thlB TTGAACC reverse CAC0462 N17CACCATGATAGTAAAAG 29 CAC0462 CAAAGTTTG forward CAC0462 N21GCTTAAAGCTTAAAACC 30 CAC0462 GCTTCTGGCG reverse ald N27F1CACCATGAATAAAGACA 31 ald forward CACTAATACC ald N28R1 GCCAGACCATCTTTGAA32 ald reverse AATGCGC thlA N44 CATGCATGCAAAGGAGG 33 thlATTAGTAGAATGAAAGAAG forward thlA N45 GTCCTGCAGGGCGCGC 34 thlACCAATACTTTCTAGCAC reverse TTTTC hbd N42 CATGTCGACAAAGGAGG 35 hbdTCTGTTTAATGAAAAAG forward GTATG hbd N43 GTCGCATGCCTTGTAAA 36 hbdCTTATTTTGAA reverse CAC0462 N68 CATAGATCTGGATCCAA 37 CAC0462AGGAGGGTGAGGAAAT forward GATAGTAAAAG CAC0462 N69 CATGTCGACGTGCAGCC 38CAC0462 TTTTTAAGGTTCT reverse crt N38 CATGAATTCACGCGTAA 39 crt forwardAGGAGGTATTAGTCATG GAAC crt N39 GTCGGATCCCTTACCTC 40 crt reverseCTATCTATTTTTG ald N58 CATGCCCGGGGGTCAC 41 ald forward CAAAGGAGGAATAGTTCATGAATAAA ald N59 CATGGTTAACAAGAAGT 42 ald reverse TAGCCGGCAAGTACA bdhBN64 CATGGTTAACAAAGGAG 43 bdhB GGGTTAAAATGGTTGAT forward TTCGAAT bdhB N65CATGGCATGCGTTTAAA 44 bdhB CGTAGGTTTACACAGAT reverse TTT — BenFACTTTCTTTCGCCTGTTT 73 — CAC — BenMAR CATGAAGCTTGGCGCG 74 —CCGGGACGCGTTTTTGA AAATAATGAAAACT — BenBPR CATGAAGCTTGTTTAAA 75 —CTCGGTGACCTTGAAAA TAATGAAAACTTATATTG TTTTGAAAATAATGAAAA CTTATATTG EgTERN85 CATAGATCTGGATCCAA 80 Egter (opt) AGGAGGGTGAGGAAAT forwardGGCGATGTTTACG EgTER N86 GTCGACTTACTGCTGGG 81 Egter (opt) CGG reversePtrc-ald T-Ptrc TTCCGTACTTCCGGACG 87 Ptrc (opt) (BspEI)ACTGCACGGTGCACCAA forward TGCTTCTG Ptrc-ald B-aldopt CGGATCTTAAGTACTTT88 ald reverse (opt) (Scat) AAC CCGCCAGCACACAGCG GCGCTGG ald AFCATTGGATCCATGAATA 93 ald forward BamHI AAGACACACTAATACCT ACAAC aldAR Aat2 CATGACGTCACTAGTGT 94 ald reverse TAACAAGAAGTTAGCCG GCAAG EgTERForward CATGTTAACAAAGGAGG 95 EgTER 1 (E) AAAGATCTATGGCGATG SOETTTACGACCACCGCAA forward EgTER Bottom CCCCTCCTTTGGCGCGC 96 EgTER ReverseCTTACTGCTGGGCGGC SOE I (E) GCTCGGCAGA reverse bdh Top GCCCAGCAGTAAGGCG97 bdh SOE Forward CGCCAAAGGAGGGGTT forward 2 (B) AAAATGGTTGATTTCGA ATbdh Reverse GTCGACGTCATACTAGT 98 bdh SOE 2 (B) TTACACAGATTTTTTGAAreverse TATTTGT — Pamy/ CATTGTACAGAATTCGA 99 Pamy lacO FGCTCTCGAGGCCCCGC forward ACATACGAAAAGAC — Pamy/ CATTGTACAGTTTAAACA 100Pamy lacO R TAGGTCACCCTCATTTT reverse CGTAGGAATTGTTATCC — Spac FCATCTCGAGTAATTCTA 101 Pspac CACAGCCCAGTCC forward — Spac RCATGTTTAAACGGTGAC 102 Pspac CCAAGCTGGGGATCCG reverse CGG thl Top TFCATTGGTCACCATTCCC 103 thl SOE GGGCATGCAAAGGAGG Forward TTAGTAGAATG thlBot TR CCTTTACGCGACCGGTA 104 thl SOE CTAGTCAAGTCGACAGG reverseGCGCGCCCAATACTTTC crt Top CF CGCGCCCTGTCGACTTG 105 crt SOEACTAGTACCGGTCGCGT forward AAAGGAGGTATTAGTCA TGGAAC crt Bot CRCATCGTTTAAACTTGGA 106 crt SOE TCCAGATCCCTTACCTC reverse CTAT ERG10-OT731 AAAGCTGGAGCTCCACC 164 ERG10- ERG10t GCGGTGGCGGCCGCTC ERG10tTAGAAGTTTTCAAAGCA forward GAGTTTCGTTTGAATATT TTACCA ERG10- OT732TTCAATATGCATGCCTC 165 ERG10- ERG10t AGAACGTTTACATTGTAT ERG10tCGACTGCCAGAACCC reverse GAL1- OT733 GCAGTCGATACAATGTA 166 GAL1- GAL10AACGTTCTGAGGCATGC GAL10 ATATTGAATTTTCAAAAA forward TTCTTACTTTTTTTTTGGATGGACGCA GAL1- OT734 ACCTGCACCTATAACAC 167 GAL1- GAL10ATACCTTTTCCATGGTA GAL10 GTTTTTTCTCCTTGACGT reverse TAAAGTATAGAGGTATA TTAhbd OT735 AAAAACTACCATGGAAA 168 hbd AGGTATGTGTTATAGGT forwardGCAGGTACTATGGGTTC AGGAATTGC hbd OT736 GTAAAAAAAAGAAGGCC 169 hbdGTATAGGCCTTATTTTG reverse AATAATCGTAGAAACCT TTTCCTGATTTTCTTCCA AG GAL1tOT737 ACGATTATTCAAAATAAG 170 GAL1t GCCTATACGGCCTTCTT forwardTTTTTTACTTTGTTCAGA ACAACTTCTCATTTTTTT CTACTCATAA GAL1t OT738GAATTGGGTACCGGGC 171 GAL1t CCCCCCTCGAGGTCGA reverse CCGATGCCTCATAAACTTCGGTAGTTATATTACTC TGAGAT thlA OT797 AAAGTAAGAATTTTTGAA 172 thlAAATTCAATATGCATGCA forward AGAAGTTGTAATAGCTA GTGCAGTAAGAAC thlA OT798GAAAAAGATCATGAGAA 173 thlA AATCGCAGAACGTAAGG reverse CGCGCCTCAGCACTTTTCTAGCAATATTGCTGTT CCTTG CUP1 OT806  CTCGAAAATAGGGCGC 174 CUP1GCCCCCATTACCGACAT forward TTGGGCGC CUP1 OT807  ACTGCACTAGCTATTAC 175CUP1 AACTTCTTGCATGCGTG reverse ATGATTGATTGATTGATT GTA GPD OT808TCGGTAATGGGGGCGC 176 GPD promoter GCCCTATTTTCGAGGAC promoterCTTGTCACCTTGA forward GPD OT809 TTTCGAATAAACACACAT 177 GPD promoterAAACAAACACCCCATGG promoter AAAAGGTATGTGTTATA reverse GGTGCAGG FBA1 OT799TACCGGGCCCCCCCTC 178 FBA1 promoter GAGGTCGACGGCGCGC promoterCACTGGTAGAGAGCGA forward CTTTGTATGCCCCA FBA1 OT761 CTTGGCCTTCACTAGCA 179FBA1 promoter TGCTGAATATGTATTACT promoter TGGTTATGGTTATATATG reverseACAAAAG GPM1 OT803 CCCTCACTAAAGGGAAC 180 GPM1 promoter AAAAGCTGGAGCTCGATpromoter ATCGGCGCGCCCACAT forward GCAGTGATGCACGCGC GA GPM1 OT804AAGGATGACATTGTTTA 181 GPM1 promoter GTTCCATGGTTGTAATA promoterTGTGTGTTTGTTTGG reverse crt OT785 CACACATATTACAACCA 182 Crt forwardTGGAACTAAACAATGTC ATCCTTGAAAAGGAAGG crt OT786 ATCATTCATTGGCCATT 183Crt reverse CAGGCCTTATCTATTTTT GAAGCCTTCAATTTTTCT TTTCTCTATG GPM1t OT787CAAAAATAGATAAGGCC 184 GPM1t terminator TGAATGGCCAATGAATG terminatorATTTGATGATTTCTTTTT forward CCCTCCATTTTTC GPM1t OT805 GAATTGGGTACCGGGC185 GPM1t terminator CCCCCCTCGAGGTCGA terminator CTTATAGTATTATATTTTreverse CTGATTTGGTTATAGCA AGCAGCGTTT GPD OT800 GGGAACAAAAGCTGGA 190 GPDpromoter GCTCCACCGCGGTGGG promoter GCGCGCCCTATTTTCGA forwardGGACCTTGTCACCTTGA GCC GPD OT758 TTAAGGTATCTTTATCCA 191 GPD promoterTGGTGTTTGTTTATGTGT promoter GTTTATTCGAAACT reverse GPD OT754TTGGGTACCGGGCCCC 192 GPD terminator CCCTCGAGGTCGACTG terminatorGCCATTAATCTTTCCCAT forward AT GPD OT755 TGTGTCCTAGCAGGTTA 193 GPDterminator GGGCCTGCAGGGCCGT terminator GAATTTACTTTAAATCTTG reverse FBA1OT760 CGAAAATAGGGCGCGC 194 FBA1 promoter CACTGGTAGAGAGCGA promoterCTTTGTATGCCCCAATTG forward FBA1 OT792 CCCTTGACGAACTTGGC 195 FBA1promoter CTTCACTAGCATGCTGA promoter ATATGTATTACTTGGTTA reverseTGGTTATATATGACAAAAG FBA1 OT791 CCCTTGACGAACTTGGC 196 FBA1 terminatorCTTCACTAGCATGCTGA terminator ATATGTATTACTTGGTTA forwardTGGTTATATATGACAAAAG FBA1 OT765 GGAACAAAAGCTGGAG 197 FBA1 terminatorCTCCACCGCGGTGGTTT terminator AACGTATAGACTTCTAAT reverseATATTTCTCCATACTTGG TATT ldhL LDH GACGTCATGACCACCCG 198 ldhL EcoRVCCGATCCCTTTT forward F ldhL LDH GATATCCAACACCAGCG 199 ldhL AatIIRACCGACGTATTAC reverse Cm Cm F ATTTAAATCTCGAGTAG 200 Cm AGGATCCCAACAAACGAforward AAATTGGATAAAG Cm Cm R ACGCGTTATTATAAAAG 201 Cm CCAGTCATTAGGreverse P11 P11 F TCGAGAGCGCTATAGTT 202 P11 GTTGACAGAATGGACAT promoterACTATGATATATTGTTGC forward TATAGCGCCC P11 P11 R GGGCGCTATAGCAACAA 203P11 TATATCATAGTATGTCCA promoter TTCTGTCAACAACTATA reverse GCGCTC PldhLPldhL F GAGCTCGTCGACAAACC 204 ldhL AACATTATGACGTGTCT promoter GGGCforward PldhL PldhL R GGATCCTACCATGTTTG 205 ldhL TGCAAAATAAGTG promoterreverse PnisA F-PnisA TTCAGTGATATCGACAT 206 PnisA (EcoRV)ACTTGAATGACCTAGTC forward PnisA R-PnisA TTGATTAGTTTAAACTGT 207 PnisA(Pmel AGGATCCTTTGAGTGCC reverse BamHI) TCCTTATAATTTA

TABLE 5  Sequencing and PCR Screening Primers Gene- SEQ ID Name Sequencespecific NO: M13 Forward GTAAAACGACGGCCAGT TOPO 45 vector M13 ReverseAACAGCTATGACCATG TOPO 46 vector N7SeqF1 GCAGGAGATGCTGACGTAATAA thlA 47N7SeqR1 CCAACCTGCTTTTTCAATAGCTGC thlA 48 N15SegF1 CAGAGATGGGGTCAAAGAATGthlB 49 N16SeqR1 GTGGTTTTATTCCGAGAGCG thlB 50 N5SeqF2GGTCTATACTTAGAATCTCC hbd 51 N6SeqR2 CGGAACAGTTGACCTTAATATGGC hbd 52N22SeqF1 GCCTCATCTGGGTTTGGTCTTG CAC0426 53 N22SeqF2CGCCTAGGAGAAAGGACTATAA CAC0426 54 AACTGG N22SeqF3 CAGAGTTATAGGTGGTAGAGCCCAC0426 55 N23SeqR1 CCATCCCGCTGTTCCTATTCTTCT CAC0426 56 N23SeqR2CCAATCCTCTCCACCCATTACC CAC0426 57 N23SeqR3 CGTCCATCCTTAATCTTCCC CAC042658 N31SeqF2 CCAACTATGGAATCCCTAGATGC ald 59 N31SeqF3GCATAGTCTGCGAAGTAAATGC ald 60 N31SeqF4 GGATCTACTGGTGAAGGCATAACC ald 61N32SeqR1 GTTAGCCGGCAAGTACACATC ald 72 N32SeqR2 GGCATCATGAGTTCTGTCATGACald 62 N32SeqR3 GCCTTCAATGATACTCTTACCAGCC ald 63 N32SeqR4GCATTTCCAGCAGCTATCATGC ald 64 N32SeqR5 CCTTCCCATATGTGTTTCTTCC ald 65N11SeqF1 GTTGAAGTAGTACTAGCTATAG bdhB 66 N11 SeqF2 GACATAACACACGGCGTAGGGCbdhB 67 N12SeqR1 TAAGTGTACACTCCAATTAGTG bdhB 68 N12SeqR2GCCATCTAACACAATATCCCATGG bdhB 69 N9SeqF1 GCGATACATGGGACATGGTTAAAG bdhA70 N10SeqR1 TGCACTTAACTCGTGTTCCATA bdhA 71 T7Primer TAATACGACTCACTATAGGGpET23 82 vector Trc99aF TTGACAATTAATCATCCGGC p Trc99a 83 vector N5SeqF4GGTCAACTGTTCCGGAAATTC hbd 84 T-ald(BamHI) TGATCTGGATCCAAGAAGGAGC ald 85CCTTCACCATGAATAAAGACACAC B-ald(EgTER) CATCGCCATTTCCTCACCCTCCT ald 86TTTTAGCCGGCAAGTACACATCT TCTTTGTC N3SeqF1 CCATCATACCATACTGACCC crt 107N3SeqF2 GCTACTGGAGCATTGCTCAC crt 108 N3SeqF3 CCATTAACAGCTGCTATTACAGGCcrt 109 N4SeqR3 GGTCTCGGAATAACACCTGG crt 110 N5SeqF3CAAGCTTCATAACAGGAGCTGG hbd 111 N7SeqR2 ATCCCACAATCCGTCAGTGATC thlA 112N31SeqF1 CTGAGATAAGAAAGGCCGCA ald 113 N62SeqF2 CAACCCTGGGCGTGTTTCTGEgTER 114 N62SeqF3 GTGGCGAAGATTGGGAACTG EgTER 115 N62SeqF4GGGAAATGGCAGAAGATGTTCAGC EgTER 116 N63SeqR1 CGGTCTGATAACCTGCAAAATCGCEgTER 117 N63SeqR2 CACCAGCGCTTTGGCAACAAC EgTER 118 N63SeqR3GAACGTGCATACAGACCTGCTTC EgTER 119 N63SeqR4 CGGCTGAATAACTTTTGCGG EgTER120 Pamy SeqF2 GCCTTTGATGACTGATGATTTGGC pFP988 121 vector Pamy SeqFTCTCCGGTAAACATTACGGCAAAC pFP988 122 vector Pamy SeqRCGGTCAGATGCAATTCGACATGTG pFP988 123 vector SpacF SeqGAAGTGGTCAAGACCTCACT Pspac 124 promoter sacB Up CGGGTTTGTTACTGATAAAGCAGGsacB 125 sacB Dn CGGTTAGCCATTTGCCTGCTTTTA sacB 126 HT RACAAAGATCTCCATGGACGCGT pHT01 127 vector Scr1 CCTTTCTTTGTGAATCGG csc 160Scr2 AGAAACAGGGTGTGATCC csc 161 Scr3 AGTGATCATCACCTGTTGCC csc 162 Scr4AGCACGGCGAGAGTCGACGG csc 163

Methods for Determining 1-Butanol Concentration in Culture Media

The concentration of 1-butanol in the culture media can be determined bya number of methods known in the art. For example, a specific highperformance liquid chromatography (HPLC) method utilized a ShodexSH-1011 column with a Shodex SH-G guard column, both purchased fromWaters Corporation (Milford, Mass.), with refractive index (RI)detection. Chromatographic separation was achieved using 0.01 M H₂SO₄ asthe mobile phase with a flow rate of 0.5 mL/min and a column temperatureof 50° C. 1-Butanol had a retention time of 52.8 min under theconditions used. Alternatively, gas chromatography (GC) methods areavailable. For example, a specific GC method utilized an HP-INNOWaxcolumn (30 m×0.53 mm id, 1 μm film thickness, Agilent Technologies,Wilmington, Del.), with a flame ionization detector (FID). The carriergas was helium at a flow rate of 4.5 mL/min, measured at 150° C. withconstant head pressure; injector split was 1:25 at 200° C.; oventemperature was 45° C. for 1 min, 45 to 220° C. at 10° C./min, and 220°C. for 5 min; and FID detection was employed at 240° C. with 26 mL/minhelium makeup gas. The retention time of 1-butanol was 5.4 min. Asimilar GC method using a Varian CP-WAX 58(FFAP) CB column (25 m×0.25 mmid×0.2 μm film thickness, Varian, Inc., Palo Alto, Calif.) was alsoused.

The meaning of abbreviations is as follows: “s” means second(s), “min”means minute(s), “h” means hour(s), “psi” means pounds per square inch,“nm” means nanometers, “d” means day(s), “μL” means microliter(s), “mL”means milliliter(s), “L” means liter(s), “mm” means millimeter(s), “nm”means nanometers, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” means micromole(s)”, “g” means gram(s), “μg” meansmicrogram(s) and “ng” means nanogram(s), “PCR” means polymerase chainreaction, “OD” means optical density, “OD₆₀₀” means the optical densitymeasured at a wavelength of 600 nm, OD₅₅₀″ means the optical densitymeasured at a wavelength of 550 nm, “kDa” means kilodaltons, “g” meansthe gravitation constant, “rpm” means revolutions per minute, “bp” meansbase pair(s), “kbp” means kilobase pair(s), “% w/v” means weight/volumepercent, % v/v″ means volume/volume percent, “nt” means not tested,“HPLC” means high performance liquid chromatography, and “GC” means gaschromatography.

Example 1 Increased Tolerance of Lactobacillus plantarum PN0512 to1-Butanol, Iso-Butanol and 2-Butanol at Decreased Growth Temperatures

Tolerance levels of bacterial strain Lactobacillus plantarum PN0512(ATCC #PTA-7727) were determined at 25° C., 30° C. and 37° C. asfollows. The strain was cultured in S30L medium (i.e., 10 mM ammoniumsulfate, 5 mM potassium phosphate buffer, pH 7.0, 50 mM MOPS, pH 7.0, 2mM MgCl₂, 0.7 mM CaCl₂, 50 μM MnCl₂, 1 μM FeCl₃, 1 μM ZnCl₂, 1.72 μMCuCl₂, 2.53 μM

CoCl₂, 2.42 μM Na₂MoO₄, 2 μM thiamine hydrochloride, 10 mM glucose, and0.2% yeast extract). An overnight culture in the absence of any testcompound was started in 15 mL of the S30L medium in a 150 mL flask, withincubation at 37° C. in a shaking water bath. The next morning, theovernight culture was diluted into three 500 mL flasks containing 150 mLof fresh medium to an initial OD₆₀₀ of about 0.08. Each flask wasincubated in a shaking water bath, one each at 25° C., 30° C. and 37° C.Each large culture was allowed to acclimate at the test temperature forat least 0.5 h. After the acclimation period, each large culture wassplit into flasks in the absence (control) and in the presence ofvarious amounts of 1-butanol, isobutanol or 2-butanol, as listed inTables 6, 7, and 8, respectively. Growth was followed by measuring OD₆₀₀for six hours after addition of the compounds. The results aresummarized in Tables 6, 7, and 8 below.

TABLE 6 Growth of L. plantarum PN0512 in the presence of 1-butanol atdifferent temperatures Concentration 1- butanol (% w/v) 37° C. 30° C.25° C. 0.0 + + + 1.0 + nt nt 1.2 + nt nt 1.4 + nt nt 1.5 + + + 1.6 + ntnt 1.8 + nt nt 2.0 + + + 2.1 + nt nt 2.2 + nt nt 2.3 + nt nt 2.4 − + +2.5 − nt nt 2.7 − + nt 2.9 − − + 3.1 − − + 3.2 nt − − 3.3 nt nt − 3.4 nt− −

TABLE 7 Growth of L. plantarum PN0512 in the presence of isobutanol atdifferent temperatures Concentration isobutanol (% w/v) 37° C. 30° C.25° C. 0.0 + + + 0.5 + nt nt 1.0 + nt nt 1.5 + + + 1.6 + nt nt 1.8 + ntnt 2.0 + + + 2.1 + nt nt 2.3 + nt nt 2.4 + + + 2.5 + nt nt 2.7 + + +2.9 + + + 3.1 + + + 3.3 nt − + 3.4 − nt nt 3.5 nt nt + 3.6 nt nt − 3.8 −nt nt 4.3 − nt nt

TABLE 8 Growth of L. plantarum PN0512 in the presence of 2-butanol atdifferent temperatures Concentration 2- butanol (% w/v) 37° C. 30° C.25° C. 0.0 + + + 1.8 + nt nt 2.1 + nt nt 2.5 + nt nt 2.9 + + + 3.1 + ntnt 3.5 + nt nt 3.6 + nt nt 3.8 + + + 4.0 nt + nt 4.3 + + + 4.5 − + nt4.7 − + + 4.9 nt − + 5.2 − nt + 5.6 − nt − 6.0 − nt nt 6.4 − nt nt 7.3 −nt nt “+” = growth observed as an increase in OD₆₀₀. “−” = no growthobserved, i.e. no change in OD₆₀₀.

All three butanols showed a similar effect of temperature on growthinhibition of L. plantarum PN0512. The concentration that resulted infull growth inhibition was greater at 25° C. than at 37° C. In the caseof 1-butanol, growth was observed at 37° C. in 2.3% 1-butanol, but not2.4%. However, at 30° C. growth was observed in 2.7%, but not 2.9%, andat 25° C. growth was observed even in 3.1% 1-butanol. Thus, theconcentration of 1-butanol that completely inhibited growth increased asgrowth temperature decreased. Likewise, in the case of isobutanol,growth was observed in 3.5% at 25° C. while growth was observed in 3.1%at 30° C. and 37° C., but not in 3.3% or 3.4%. Similarly, in the case of2-butanol growth was observed at 37° C. in 4.3%, but not in 4.5%; at 30°C. in 4.7%, but not in 4.9%; and at 25° C. in 5.2%. Thus the toleranceof L. plantarum PN0512 to butanols increased with decreased growthtemperature.

Example 2 Increased Tolerance of Escherichia coli to 1-Butanol atDecreased Exposure Temperature

The effect of growth and exposure temperature on survival of Escherichiacoli in the presence of 1-butanol was tested using stationary phasecultures in a rich medium and log phase cultures in a defined medium.For the stationary phase studies, E. coli strain MG1655 (ATCC #700926)was grown overnight in LB medium (Teknova, Half Moon Bay, Calif.) withshaking at 250 rpm at 42° C., 29° C. or 28° C. Survival of 1-butanolshock was tested at exposure temperatures of 0° C., 28° C. or 42° C. The1-butanol exposure at 28° C. or 42° C. was started immediately afterremoving the overnight cultures from the growth incubators. The1-butanol exposure at 0° C. was done after allowing the overnightcultures to cool on ice for about 15 min. A series of solutions of1-butanol at different concentrations in LB medium was made and 90 μLaliquots were put in microfuge tubes. To these were added 10 μL of theovernight cultures and the tubes were immediately placed in shakingincubators at 42° C. or 28° C. or left on ice for 30 min. To stop theeffect of 1-butanol on the cultures, a 10⁻² dilution was done by placing2 μL of the treated culture into 198 μL of LB medium in wells of amicroplate. Then, 5 μL of the undiluted treated cultures were spotted onLB agar plates. Subsequent 10-fold serial dilutions of 10⁻³, 10⁻⁴, 10⁻⁵and 10⁻⁶ of the exposed cultures were done by serial pipetting of 20μL,starting with the 10⁻² dilution cultures, into 180 μL of LB medium inthe microplate, using a multi-channel pipette. Prior to each transfer,the cultures were mixed by pipetting up and down six times. Eachdilution (5 μL) was spotted onto an LB plate using a multi-channelpipette and allowed to soak into the plate. The plates were inverted andincubated overnight at 37° C. The number of colonies for each dilutionwas counted and the % growth inhibition was calculated by comparisonwith a control culture that had not been exposed to 1-butanol. Survivalof 0% was recorded when no colonies in the spots of the undiluted or anyof the serial dilutions were observed. The results are shown in Table 9.

TABLE 9 Survival of stationary phase E. coli in 1-butanol at 42° C., 28°C., or 0° C. Grown at Grown at Grown at Grown at Grown at Grown at 42°C. 29° C. 42° C. 28° C. 42° C. 29° C. % survival after % survival after% survival after 1-Butanol 30 min exposure 30 min exposure 30 minexposure % (w/v) at 42° C. at 28° C. at 0° C. 1.0 100 100 100 100 100100 1.5 0.1 0.1 100 100 100 100 2.0 0 0.1 100 100 100 100 2.5 0 0 100100 100 100 3.0 0 0 100 100 100 100 3.5 0 0 3 10 100 100 4.0 0 0 0.00040.0003 100 100 5.0 nt nt nt nt 1 1 6.0 nt nt nt nt 0 0.001 7.0 nt nt ntnt 0 0

A similar study was done with log-phase cultures of E. coli grown in adefined medium. E. coli strain MG1655 was allowed to grow overnight inMOPS 0.2% glucose medium (Teknova, Half Moon Bay, Calif.) at 42° C. or28° C. The following day, the cultures were diluted into fresh mediumand allowed to grow at the same temperature until in the log phase ofgrowth. The OD₆₀₀ was 0.74 for the 28° C. culture and was 0.72 for the42° C. culture. Both of these log phase cultures were exposed to1-butanol at 42° C., 28° C. and 0° C. as follows. A series of solutionsof 1-butanol at different concentrations in MOPS 0.2% glucose medium wasmade and 90 μL aliquots were put in microfuge tubes. To these were added10 μL of the log phase cultures and the tubes were immediately placed inshaking incubators at 42° C. or 28° C. or left on ice for 30 min. Tostop the effect of 1-butanol on the cultures, a 10⁻² dilution was doneby placing 2 μL of the treated culture into 198 μL of LB medium in wellsof a microplate. Then 5 μL of the undiluted treated cultures werespotted on LB agar plates. Subsequent 10-fold serial dilutions of 10⁻³,10⁻⁴, 10⁻⁵ and 10⁻⁶ of the exposed cultures were done by serialpipetting of 20 μL, starting with the 10⁻² dilution cultures, into 180of LB medium in the microplate, using a multi-channel pipette. Prior toeach transfer, the cultures were mixed by pipetting up and down sixtimes. Each dilution (5 μL) was spotted onto an LB plate using amulti-channel pipette and allowed to soak into the plate. The plateswere inverted and incubated overnight at 37° C. The number of coloniesfor each dilution was counted and the % growth inhibition was calculatedby comparison with a control culture that had not been exposed to1-butanol. Survival of 0% was recorded when no colonies in the spots ofthe undiluted or any of the serial dilutions were observed. The resultsare shown in Table 10.

TABLE 10 Survival of log-phase E. coli in 1-butanol at 42° C., 28° C.,or 0° C. Grown at Grown at Grown at Grown at Grown at Grown at 42° C.28° C. 42° C. 28° C. 42° C. 29° C. % survival after % survival after %survival after 1-Butanol 30 min exposure 30 min exposure 30 min exposure% (w/v) at 42° C. at 28° C. at 0° C. 1.0 100 100 nt nt nt nt 1.5 0 0 100100 nt nt 2.0 0 0 100 100 nt nt 2.5 0 0 0.1 50 100 100 3.0 0 0 0 0 100100 3.5 0 0 0.01 0 100 100 4.0 0 0 0.001 0 100 100 4.5 nt nt 0 0 100 1005.0 nt nt nt nt 10 50 6.0 nt nt nt nt 1 1

For both the stationary phase and log-phase cultures of E. coli MG1655,the growth temperature had very little, if any, effect on the survivalof a 1-butanol shock. However, the exposure temperature had a majoreffect on the survival of E. coli to 1-butanol shock. As can be seenfrom the data in Tables 9 and 10, the tolerance of E. coli MG1655 to1-butanol increased with decreasing exposure temperature.

Example 3 Increased Tolerance of Escherichia coli to 2-Butanone atDecreased Exposure Temperature

The effect of exposure temperature on survival of Escherichia coli inthe presence of 2-butanone (also referred to herein as methyl ethylketone or MEK) was tested as follows. E. coli strain BW25113 (The ColiGenetic Stock Center (CGSC), Yale University; #7636) was grown overnightin LB medium (Teknova, Half Moon Bay, Calif.) with shaking at 250 rpm at37° C. Survival of MEK shock was tested at exposure temperatures of 28°C. or 37° C. A series of solutions of MEK at different concentrations inLB medium was made and 90 μL aliquots were put in microfuge tubes. Tothese were added 10 μL of the overnight culture and the tubes wereimmediately placed in shaking incubators at 37° C. or 28° C. for 30 min.To stop the effect of MEK on the cultures, a 10⁻² dilution was done byplacing 2 μL of the MEK treated culture into 198 μL of LB medium inwells of a microplate. Then 5 μL of the undiluted treated cultures werespotted on LB agar plates. Subsequent 10-fold serial dilutions of 10⁻³,10⁻⁴, 10⁻⁵ and 10⁻⁶ of the exposed cultures were done by serialpipetting of 20 μL, starting with the 10⁻² dilution cultures, into 180μL of LB medium in the microplate, using a multi-channel pipette. Priorto each transfer, the cultures were mixed by pipetting up and down sixtimes. Each dilution (5 μL) was spotted onto LB plates using amulti-channel pipette and allowed to soak into the plate. The plateswere inverted and incubated overnight at 37° C. The number of coloniesfor each dilution was counted and the % growth inhibition was calculatedby comparison with a control culture that had not been exposed to MEK.Survival of 0% was recorded when no colonies in the spots of theundiluted or any of the serial dilutions were observed. The results,given as the average of duplicate experiments, are shown in Table 11.

TABLE 11 Survival of E. coli in MEK at 37° C. and 28° C. MEK % w/v %Survival at 37° C. % Survival at 28° C. 0 100 100 4 100 100 6 0 100 8 00.002

Reducing the exposure temperature from 37° C. to 28° C. dramaticallyimproved survival of E. coli to MEK treatment. At 37° C. there was fullsurvival at 4% w/v and no survival at 6% w/v, while at 28° C. there wasfull survival at 6% w/v. Thus, the tolerance of E. coli to MEK increasedwith decreasing exposure temperature.

Example 4 Increased Tolerance of E. Coli and L. Plantarum PN0512 to1-Butanol at Decreased Exposure Temperature

This Example demonstrates that the toxic effects of 1-butanol and2-butanol on various microbial cells was reduced at lower temperatures.This was demonstrated by incubating E. coli (strain MG1655; ATCC#700926), and L. plantarum (strain PN0512; ATCC #PTA-7727) with either1-butanol or 2-butanol at different temperatures and then determiningthe fraction of the cells that survived the treatment at the differenttemperatures.

Using overnight cultures or cells from plates, 30 mL cultures of themicroorganisms to be tested were started in the following culture media:

-   -   E. coli—Miller's LB medium (Teknova, Half Moon Bay, Calif.):    -   L. plantarum PN0512—Lactobacilli MRS Broth (BD Diagnostic        Systems, Sparks, Md.).        The E. coli and L. plantarum cultures were grown at 37° C.        aerobically with shaking until the cultures were in log phase        and the OD₆₀₀ was between 0.6 and 0.8. A 50 μL aliquot of each        culture was removed for a time zero sample. The remainder of the        cultures was divided into six 5 mL portions and placed in six        small incubation flasks or tubes. Different amounts of 1-butanol        or 2-butanol were added to the six flasks to bring the        concentration to predetermined values, as listed in the tables        below. The flasks or tubes were incubated at a desired        temperature, aerobically without shaking for 1 h. After the        incubation with one of the butanols, 2 μL from each of the        flasks (and in addition 2 μL of the time zero sample of the        culture before exposure to one of the butanols) were pipetted        into the “head” wells of a 96 well (8×12) microtiter plate, each        containing 198 μL of LB medium to give a 10⁻² dilution of the        culture. Subsequently, 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶ serial        dilutions of the cultures were prepared as follows. The 10⁻³        dilution was prepared by pipetting 20 μL of the sample from the        head well into the 180 μL LB medium in the next well using a        multi-channel pipette. This procedure was repeated 3 more times        on successive wells to prepare the 10⁻⁴, 10⁻⁵, and 10⁻⁶        dilutions. After each liquid transfer, the solution in the well        was mixed by pipetting it up and down 10 times with the        multi-channel pipetor. A 5 μL aliquot of each dilution was        spotted onto an LB plate using a multi-channel pipette starting        with the 10⁻⁶ dilution, then the 10⁻⁶, and so on working from        more to less dilute without a change of tips. The spots were        allowed to soak into the agar by leaving the lid of the plate        slightly open for 15 to 30 min in a sterile transfer hood. The        plates were covered, inverted, and incubated overnight at 37° C.        The following day, the number of colonies in the spots were        counted from the different dilutions. The number of living        cells/mL in each of the original culture solutions from which        the 2 μL was withdrawn was calculated and compared to the number        of cells in the control untreated culture to determine the % of        the cells surviving.

The results of experiments in which E. coli cells were treated with1-butanol at temperatures of 0, 30, and 37° C. are shown Table 12.

TABLE 12 Percentage of E. coli cells surviving in 1-butanol at 0, 30 and37° C. 1-butanol % Survival at % Survival at % Survival at % v/v 0° C.30° C. 37° C. 0 100 100 100 1 nt 100 72 1.5 nt 100 20 2 nt 100 0 2.5 10023 0 3 100 0 0 3.5 100 0 nt 4 100 nt nt 4.5 100 nt nt

The concentration at which 1-butanol kills E. coli cells was affected bythe treatment temperature. At 0° C., concentrations of 1-butanol as highas 4.5% v/v had no toxic effect on E. coli cells during a one hourtreatment. At 30° C., E. coli cells were killed when treated with 3% v/v1-butanol for one hour. At 37° C., E. coli cells were killed whentreated with 2% v/v 1-butanol for one hour.

The results of experiments in which L. plantarum PN0512 cells weretreated with 1-butanol at temperatures of 0, 23, and 37° C. for one hourare shown Table 13.

TABLE 13 Percentage of L. plantarum PN0512 cells surviving in 1-butanolat 0, 23 and 37° C. 1-butanol % Survival at % Survival at % Survival at% v/v 0° C. 23° C. 37° C. 0 100 100 100 1 nt nt 80 1.5 nt nt 58 2 nt 10029 2.5 nt 100 8 3 100 82 0 3.5 100 0 0 4 100 0 nt 4.5 100 0 nt 5 0 nt nt5.5 0 nt nt

The concentration at which 1-butanol kills L. plantarum PN0512 cells wasaffected by the treatment temperature. At 0° C., concentrations of1-butanol as high as 4.5% v/v had no toxic effect on L. plantarum PN0512cells during a one hour treatment. At 23° C., L. plantarum PN0512 cellswere killed when treated with 3.5% v/v 1-butanol for one hour. At 37°C., L. plantarum PN0512 cells were killed when treated with 2.5% v/v1-butanol for one hour.

Example 5 Cloning and Expression of Acetyl-CoA Acetyltransferase

The purpose of this Example was to express the enzyme acetyl-CoAacetyltransferase, also referred to herein as acetoacetyl-CoA thiolase,in E. coli. The acetoacetyl-CoA thiolase gene thIA was cloned from C.acetobutylicum (ATCC 824) and expressed in E. coli. The thIA gene wasamplified from C. acetobutylicum (ATCC 824) genomic DNA using PCR,resulting in a 1.2 kbp product.

The genomic DNA from Clostridium acetobutylicum (ATCC 824) was eitherpurchased from the American Type Culture Collection (ATCC, Manassas,Va.) or was isolated from Clostridium acetobutylicum (ATCC 824)cultures, as described below.

Genomic DNA from Clostridium acetobutylicum (ATCC 824) was prepared fromanaerobically grown cultures. The Clostridium strain was grown in 10 mLof Clostridial growth medium (Lopez-Contreras et al., Appl. Env.Microbiol. 69(2), 869-877 (2003)) in stoppered and crimped 100 mL Bellcoserum bottles (Bellco Glass Inc., Vineland, N.J.) in an anaerobicchamber at 30° C. The inoculum was a single colony from a 2×YTG plate(Kishii, et al., Antimicrobial Agents & Chemotherapy, 47(1), 77-81(2003)) grown in a 2.5 L MGC AnaeroPak™ (Mitsubishi Gas Chemical AmericaInc, New York, N.Y.) at 37° C.

Genomic DNA was prepared using the Gentra Puregene® kit (Gentra Systems,Inc., Minneapolis, Minn.; catalog no. D-6000A) with modifications to themanufacturer's instruction (Wong et al., Current Microbiology, 32,349-356 (1996)). The thIA gene was amplified from Clostridiumacetobutylicum (ATCC 824) genomic DNA by PCR using primers N7 and N8(see Table 4), given as SEQ ID NOs:21 and 22, respectively. Other PCRamplification reagents were supplied in manufacturers' kits for example,Kod HiFi DNA Polymerase (Novagen Inc., Madison, Wis.; catalog no.71805-3) and used according to the manufacturer's protocol.Amplification was carried out in a DNA Thermocycler GeneAmp 9700 (PEApplied Biosystems, Foster city, CA).

For expression studies the Gateway cloning technology (Invitrogen Corp.,Carlsbad, Calif.) was used. The entry vector pENTR/SD/D-TOPO alloweddirectional cloning and provided a Shine-Dalgarno sequence for the geneof interest. The destination vector pDEST14 used a T7 promoter forexpression of the gene with no tag. The forward primer incorporated fourbases (CACC) immediately adjacent to the translational start codon toallow directional cloning into pENTR/SD/D-TOPO (Invitrogen) to generatethe plasmid pENTRSDD-TOPOthIA. The pENTR construct was transformed intoE. coli Top10 (Invitrogen) cells and plated according to manufacturer'srecommendations. Transformants were grown overnight and plasmid DNA wasprepared using the QIAprep Spin Miniprep kit (Qiagen, Valencia, Calif.;catalog no. 27106) according to manufacturer's recommendations. Cloneswere submitted for sequencing with M13 Forward and Reverse primers (seeTable 5), given as SEQ ID NOs:45 and 46, respectively, to confirm thatthe genes inserted in the correct orientation and to confirm thesequence. Additional sequencing primers, N7SeqF1 and N7SeqR1 (see Table5), given as SEQ ID NOs:47 and 48, respectively, were needed tocompletely sequence the PCR product. The nucleotide sequence of the openreading frame (ORF) for this gene and the predicted amino acid sequenceof the enzyme are given as SEQ ID NO:1 and SEQ ID NO:2, respectively.

To create an expression clone, the thIA gene was transferred to thepDEST 14 vector by recombination to generate pDEST14thIA. ThepDEST14thIA vector was transformed into BL21-AI cells. Transformantswere inoculated into LB medium supplemented with 50 μg/mL of ampicillinand grown overnight. An aliquot of the overnight culture was used toinoculate 50 mL of LB supplemented with 50 μg/mL of ampicillin. Theculture was incubated at 37° C. with shaking until the OD₆₀₀ reached0.6-0.8. The culture was split into two 25-mL cultures and arabinose wasadded to one of the flasks to a final concentration of 0.2% by weight.The negative control flask was not induced with arabinose. The flaskswere incubated for 4 h at 37° C. with shaking. Cells were harvested bycentrifugation and the cell pellets were resuspended in 50 mM MOPS, pH7.0 buffer. The cells were disrupted either by sonication or by passagethrough a French Pressure Cell. The whole cell lysate was centrifugedyielding the supernatant or cell free extract and the pellet or theinsoluble fraction. An aliquot of each fraction (whole cell lysate, cellfree extract and insoluble fraction) was resuspended in SDS (MES)loading buffer (Invitrogen), heated to 85° C. for 10 min and subjectedto SDS-PAGE analysis (NuPAGE 4-12% Bis-Tris Gel, catalog no. NP0322Box,Invitrogen). A protein of the expected molecular weight of about 41 kDa,as deduced from the nucleic acid sequence, was present in the inducedculture but not in the uninduced control.

Acetoacetyl-CoA thiolase activity in the cell free extracts was measuredas degradation of a Mg²⁺-acetoacetyl-CoA complex by monitoring thedecrease in absorbance at 303 nm. Standard assay conditions were 100 mMTris-HCl pH 8.0, 1 mM DTT (dithiothreitol) and 10 mM MgCl₂. The cocktailwas equilibrated for 5 min at 37° C.; then the cell-free extract wasadded. The reaction was initiated with the addition of 0.05 mMacetoacetyl-CoA plus 0.2 mM CoA. Protein concentration was measured byeither the Bradford method or by the Bicinchoninic Kit (Sigma, catalogno. BCA-1). Bovine serum albumin (Bio-Rad, Hercules, Calif.) was used asthe standard in both cases. In one typical assay, the specific activityof the ThIA protein in the induced culture was determined to be 16.0μmol mg⁻¹ min⁻¹ compared to 0.27 μmol mg⁻¹ min⁻¹ in the uninducedculture.

Example 6 Cloning and Expression of Acetyl-CoA Acetyltransferase

The purpose of this Example was to express the enzyme acetyl-CoAacetyltransferase, also referred to herein as acetoacetyl-CoA thiolase,in E. coli. The acetoacetyl-CoA thiolase gene thIB was cloned from C.acetobutylicum (ATCC 824) and expressed in E. coli. The thIB gene wasamplified from C. acetobutylicum (ATCC 824) genomic DNA using PCR.

The thIB gene was cloned and expressed in the same manner as the thIAgene described in Example 5. The C. acetobutylicum (ATCC 824) genomicDNA was amplified by PCR using primers N15 and N16 (see Table 4), givenas SEQ ID NOs:27 and 28, respectively, creating a 1.2 kbp product. Theforward primer incorporated four bases (CCAC) immediately adjacent tothe translational start codon to allow directional cloning intopENTR/SD/D-TOPO (Invitrogen) to generate the plasmid pENTRSDD-TOPOthIB.Clones were submitted for sequencing with M13 Forward and Reverseprimers, given as SEQ ID NOs:45 and 46 respectively, to confirm that thegenes inserted in the correct orientation and to confirm the sequence.Additional sequencing primers, N15SeqF1 and N16SeqR1 (see Table 5),given as SEQ ID NOs:49 and 50 respectively, were needed to completelysequence the PCR product. The nucleotide sequence of the open readingframe (ORF) for this gene and the predicted amino acid sequence of theenzyme are given as SEQ ID NO:3 and SEQ ID NO:4, respectively.

To create an expression clone, the thIB gene was transferred to thepDEST 14 (Invitrogen) vector by recombination to generate pDEST14thIB.The pDEST14thIB vector was transformed into BL21-AI cells and expressionfrom the T7 promoter was induced by addition of arabinose. A protein ofthe expected molecular weight of about 42 kDa, as deduced from thenucleic acid sequence, was present in the induced culture, but not inthe uninduced control. Enzyme assays were performed as described inExample 5. In one typical assay, the specific activity of the ThIBprotein in the induced culture was determined to be 14.9 μmol mg⁻¹ min⁻¹compared to 0.28 μmol mg⁻¹ min⁻¹ in the uninduced culture.

Example 7 Cloning and Expression of 3-Hydroxybutyryl-CoA Dehydrogenase

The purpose of this Example was to clone the hbd gene from C.acetobutylicum (ATCC 824) and express it in E. coli. The hbd gene wasamplified from C. acetobutylicum (ATCC 824) genomic DNA using PCR.

The hbd gene was cloned and expressed using the method described inExample 5. The hbd gene was amplified from C. acetobutylicum (ATCC 824)genomic DNA by PCR using primers N5 and N6 (see Table 4) given as SEQ IDNOs:19 and 20 respectively, creating a 881 bp product. The forwardprimer incorporated four bases (CACC) immediately adjacent to thetranslational start codon to allow directional cloning intopENTR/SD/D-TOPO (Invitrogen) to generate the plasmid pENTRSDD-TOPOhbd.Clones were submitted for sequencing with M13 Forward and Reverseprimers, given as SEQ ID NOs:45 and 46 respectively, to confirm that thegenes inserted in the correct orientation and to confirm the sequence.Additional sequencing primers, N5SeqF2 and N6SeqR2 (see Table 5), givenas SEQ ID NOs:51 and 52 respectively, were needed to completely sequencethe PCR product. The nucleotide sequence of the open reading frame (ORF)for this gene and the predicted amino acid sequence of the enzyme aregiven as SEQ ID NO:5 and SEQ ID NO:6, respectively.

To create an expression clone, the hbd gene was transferred to the pDEST14 (Invitrogen) vector by recombination to generate pDEST14hbd. ThepDEST14hbd vector was transformed into BL21-AI cells and expression fromthe T7 promoter was induced by addition of arabinose, as described inExample 5. A protein of the expected molecular weight of about 31 kDa,as deduced from the nucleic acid sequence, was present in the inducedculture, but was absent in the uninduced control.

Hydroxybutyryl-CoA dehydrogenase activity was determined by measuringthe rate of oxidation of NADH as measured by the decrease in absorbanceat 340 nm. A standard assay mixture contained 50 mM MOPS, pH 7.0, 1 mMDTT and 0.2 mM NADH. The cocktail was equilibrated for 5 min at 37° C.and then the cell free extract was added. Reactions were initiated byaddition of the substrate, 0.1 mM acetoacetyl-CoA. In one typical assay,the specific activity of the BHBD protein in the induced culture wasdetermined to be 57.4 μmol mg⁻¹ min⁻¹ compared to 0.885 μmol mg⁻¹ min⁻¹in the uninduced culture.

Example 8 Cloning and Expression of Crotonase

The purpose of this Example was to clone the crt gene from C.acetobutylicum (ATCC 824) and express it in E. coli. The crt gene wasamplified from C. acetobutylicum (ATCC 824) genomic DNA using PCR.

The crt gene was cloned and expressed using the method described inExample 5. The crt gene was amplified from C. acetobutylicum (ATCC 824)genomic DNA by PCR using primers N3 and N4 (see Table 4), given as SEQID NOs:17 and 18, respectively, creating a 794 bp product. The forwardprimer incorporated four bases (CACC) immediately adjacent to thetranslational start codon to allow directional cloning intopENTR/SD/D-TOPO (Invitrogen) to generate the plasmid pENTRSDD-TOPOcrt.Clones were submitted for sequencing with M13 Forward and Reverseprimers, given as SEQ ID NOs:45 and 46 respectively, to confirm that thegenes inserted in the correct orientation and to confirm the sequence.The nucleotide sequence of the open reading frame (ORF) for this geneand its predicted amino acid sequence are given as SEQ ID NO:7 and SEQID NO:8, respectively.

To create an expression clone, the crt gene was transferred to the pDEST14 (Invitrogen) vector by recombination to generate pDEST14crt. ThepDEST14crt vector was transformed into BL21-AI cells and expression fromthe T7 promoter was induced by addition of arabinose, as described inExample 5. A protein of the expected molecular weight of about 28 kDa,as deduced from the nucleic acid sequence, was present in much greateramounts in the induced culture than in the uninduced control.

Crotonase activity was assayed as described by Stern (Methods Enzymol.1, 559-566, (1954)). In one typical assay, the specific activity of thecrotonase protein in the induced culture was determined to be 444 μmolmg⁻¹ min⁻¹ compared to 47 μmol mg⁻¹ min⁻¹ in the uninduced culture.

Example 9 Cloning and Expression of Butyryl-CoA Dehydrogenase

The purpose of this Example was to express the enzyme butyryl-CoAdehydrogenase, also referred to herein as trans-2-Enoyl-CoA reductase,in E. coli. The CAC0462 gene, a putative trans-2-enoyl-CoA reductasehomolog, was cloned from C. acetobutylicum (ATCC 824) and expressed inE. coli. The CAC0462 gene was amplified from C. acetobutylicum (ATCC824) genomic DNA using PCR.

The CAC0462 gene was cloned and expressed using the method described inExample 5. The CAC0462 gene was amplified from C. acetobutylicum (ATCC824) genomic DNA by PCR using primers N17 and N21 (see Table 4), givenas SEQ ID NOs:29 and 30, respectively, creating a 1.3 kbp product. Theforward primer incorporated four bases (CACC) immediately adjacent tothe translational start codon to allow directional cloning intopENTR/SD/D-TOPO (Invitrogen) to generate the plasmidpENTRSDD-TOPOCAC0462. Clones were submitted for sequencing with M13Forward and Reverse primers, given as SEQ ID NO:45 and 46 respectively,to confirm that the genes inserted in the correct orientation and toconfirm the sequence. Additional sequencing primers, N22SeqF1 (SEQ IDNO:53), N22SeqF2 (SEQ ID NO:54), N22SeqF3 (SEQ ID NO:55), N23SeqR1 (SEQID NO:56), N23SeqR2 (SEQ ID NO:57), and N23SeqR3 (SEQ ID NO:58) (seeTable 5) were needed to completely sequence the PCR product. Thenucleotide sequence of the open reading frame (ORF) for this gene andthe predicted amino acid sequence of the enzyme are given as SEQ ID NO:9and SEQ ID NO:10, respectively.

To create an expression clone, the CAC0462 gene was transferred to thepDEST 14 (Invitrogen) vector by recombination to generatepDEST14CAC0462. The pDEST14CA0462 vector was transformed into BL21-AIcells and expression from the T7 promoter was induced by addition ofarabinose, as described in Example 5. Analysis by SDS-PAGE showed nooverexpressed protein of the expected molecular weight in the negativecontrol or in the induced culture. The C. acetobutylicum CAC0462 geneused many rare E. coli codons. To circumvent problems with codon usagethe pRARE plasmid (Novagen) was transformed into BL21-AI cells harboringthe pDEST14CAC0462 vector. Expression studies with arabinose inductionwere repeated with cultures carrying the pRARE vector. A protein of theexpected molecular weight of about 46 kDa was present in the inducedculture but not in the uninduced control.

Trans-2-enoyl-CoA reductase activity was assayed as described byHoffmeister et al. (J. Biol. Chem. 280, 4329-4338 (2005)). In onetypical assay, the specific activity of the TER CAC0462 protein in theinduced culture was determined to be 0.694 μmol mg⁻¹ min⁻¹ compared to0.0128 μmol mg⁻¹ min⁻¹ in the uninduced culture.

Example 10 Cloning and Expression of Butyraldehyde Dehydrogenase(Acetylating)

The purpose of this Example was to clone the ald gene from C.beijerinckii (ATCC 35702) and express it in E. coli. The ald gene wasamplified from C. beijerinckii (ATCC 35702) genomic DNA using PCR.

The ald gene was cloned and expressed using the method described inExample 5. The ald gene was amplified from C. beijerinckii (ATCC 35702)genomic DNA (prepared from anaerobically grown cultures, as described inExample 5) by PCR using primers N27 F1 and N28 R1 (see Table 4), givenas SEQ ID NOs:31 and 32 respectively, creating a 1.6 kbp product. Theforward primer incorporated four bases (CACC) immediately adjacent tothe translational start codon to allow directional cloning intopENTR/SD/D-TOPO (Invitrogen) to generate the plasmid pENTRSDD-TOPOald.Clones were submitted for sequencing with M13 Forward and Reverseprimers, given as SEQ ID NOs:45 and 46 respectively, to confirm that thegenes inserted in the correct orientation and to confirm the sequence.Additional sequencing primers, N31SeqF2 (SEQ ID NO:59), N31SeqF3 (SEQ IDNO:60), N31SeqF4 (SEQ ID NO:61), N32SeqR1 (SEQ ID NO:72), N31SeqR2 (SEQID NO:62), N31SeqR3 (SEQ ID NO:63), N31SeqR4 (SEQ ID NO:64), andN31SeqR5 (SEQ ID NO:65) (see Table 5) were needed to completely sequencethe PCR product. The nucleotide sequence of the open reading frame (ORF)for this gene and the predicted amino acid sequence of the enzyme aregiven as SEQ ID NO:11 and SEQ ID NO:12, respectively.

To create an expression clone, the ald gene was transferred to the pDEST14 (Invitrogen) vector by recombination to generate pDEST14ald. ThepDEST14ald vector was transformed into BL21-AI cells and expression fromthe T7 promoter was induced by addition of arabinose, as described inExample 5. A protein of the expected molecular weight of about 51 kDa,as deduced from the nucleic acid sequence, was present in the inducedculture, but not in the uninduced control.

Acylating aldehyde dehydrogenase activity was determined by monitoringthe formation of NADH, as measured by the increase in absorbance at 340nm, as described by Husemann et al. (Appl. Microbiol. Biotechnol.31:435-444 (1989)). In one typical assay, the specific activity of theAld protein in the induced culture was determined to be 0.106 μmol mg⁻¹min⁻¹ compared to 0.01 μmol mg⁻¹ min⁻¹ in the uninduced culture.

Example 11 Cloning and Expression of Butanol Dehydrogenase

The purpose of this Example was to clone the bdhB gene from C.acetobutylicum (ATCC 824) and express it in E. coli. The bdhB gene wasamplified from C. acetobutylicum (ATCC 824) genomic DNA using PCR.

The bdhB gene was cloned and expressed using the method described inExample 5. The bdhB gene was amplified from C. acetobutylicum (ATCC 824)genomic DNA by PCR using primers N11 and N12 (see Table 4), given as SEQID NOs:25 and 26, respectively, creating a 1.2 kbp product. The forwardprimer incorporated four bases (CACC) immediately adjacent to thetranslational start codon to allow directional cloning intopENTR/SD/D-TOPO (Invitrogen) to generate the plasmid pENTRSDD-TOPObdhB.The translational start codon was also changed from “GTG” to “ATG” bythe primer sequence. Clones were submitted for sequencing with M13Forward and Reverse primers, given as SEQ ID NOs:45 and 46 respectively,to confirm that the genes inserted in the correct orientation and toconfirm the sequence. Additional sequencing primers, N11SeqF1 (SEQ IDNO:66), N11SeqF2 (SEQ ID NO:67), N12SeqR1 (SEQ ID NO:68), and N12SeqR2(SEQ ID NO:69), (see Table 5) were needed to completely sequence the PCRproduct. The nucleotide sequence of the open reading frame (ORF) forthis gene and the predicted amino acid sequence of the enzyme are givenas SEQ ID NO:13 and SEQ ID NO:14, respectively.

To create an expression clone, the bdhB gene was transferred to thepDEST 14 (Invitrogen) vector by recombination to generate pDEST14bdhB.The pDEST14bdhB vector was transformed into BL21-AI cells and expressionfrom the T7 promoter was induced by addition of arabinose, as describedin Example 5. A protein of the expected molecular weight of about 43kDa, as deduced from the nucleic acid sequence, was present in theinduced culture, but not in the uninduced control.

Butanol dehydrogenase activity was determined from the rate of oxidationof NADH as measured by the decrease in absorbance at 340 nm as describedby Husemann and Papoutsakis, supra. In one typical assay, the specificactivity of the BdhB protein in the induced culture was determined to be0.169 μmol mg⁻¹ min⁻¹ compared to 0.022 μmol mg⁻¹ min⁻¹ in the uninducedculture.

Example 12 Cloning and Expression of Butanol Dehydrogenase

The purpose of this Example was to clone the bdhA gene from C.acetobutylicum 824 and express it in E. coli. The bdhA gene wasamplified from C. acetobutylicum 824 genomic DNA using PCR.

The bdhA gene was cloned and expressed using the method described inExample 5. The bdhA Gene was Amplified from C. Acetobutylicum 824Genomic DNA by PCR using primers N9 and N10 (see Table 4), given as SEQID NOs:23 and 24, respectively, creating a 1.2 kbp product. The forwardprimer incorporated four bases (CACC) immediately adjacent to thetranslational start codon to allow directional cloning intopENTR/SD/D-TOPO (Invitrogen) to generate the plasmid pENTRSDD-TOPObdhA.Clones, given as SEQ ID NOs:45 and 46 respectively, to confirm that thegenes inserted in the correct orientation and to confirm the sequence.Additional sequencing primers, N9SeqF1 (SEQ ID NO:70) and N10SeqR1 (SEQID NO:71), (see Table 5) were needed to completely sequence the PCRproduct. The nucleotide sequence of the open reading frame (ORF) forthis gene and the predicted amino acid sequence of the enzyme are givenas SEQ ID NO:15 and SEQ ID NO:16, respectively.

To create an expression clone, the bdhA gene was transferred to thepDEST 14 (Invitrogen) vector by recombination to generate pDEST14bdhA.The pDEST14bdhA vector was transformed into BL21-AI cells and expressionfrom the T7 promoter was induced by addition of arabinose, as describedin Example 5. A protein of the expected molecular weight of about 43kDa, as deduced from the nucleic acid sequence, was present in theinduced culture, but not in the uninduced control.

Butanol dehydrogenase activity was determined from the rate of oxidationof NADH as measured by the decrease in absorbance at 340 nm, asdescribed by Husemann and Papoutsakis, supra. In one typical assay, thespecific activity of the BdhA protein in the induced culture wasdetermined to be 0.102 μmol mg⁻¹ min⁻¹ compared to 0.028 μmol mg⁻¹ min⁻¹in the uninduced culture.

Example 13 Construction of a Transformation Vector for the Genes in the1-Butanol Biosynthetic Pathway—Lower Pathway

To construct a transformation vector comprising the genes encoding thesix steps in the 1-butanol biosynthetic pathway, the genes encoding the6 steps in the pathway were divided into two operons. The upper pathwaycomprises the first four steps catalyzed by acetyl-CoAacetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, andbutyryl-CoA dehydrogenase. The lower pathway comprises the last twosteps, catalyzed by butyraldehyde dehydrogenase and butanoldehydrogenase.

The purpose of this Example was to construct the lower pathway operon.Construction of the upper pathway operon is described in Example 14.

The individual genes were amplified by PCR with primers thatincorporated restriction sites for later cloning and the forward primerscontained an optimized E. coli ribosome binding site (AAAGGAGG). PCRproducts were TOPO cloned into the pCR 4Blunt-TOPO vector andtransformed into E. coli Top10 cells (Invitrogen). Plasmid DNA wasprepared from the TOPO clones and the sequence of the genes wasverified. Restriction enzymes and T4 DNA ligase (New England Biolabs,Beverly, Mass.) were used according to manufacturer's recommendations.For cloning experiments, restriction fragments were purified by gelelectrophoresis using QIAquick Gel Extraction kit (Qiagen).

After confirmation of the sequence, the genes were subcloned into amodified pUC19 vector as a cloning platform. The pUC19 vector wasmodified by a HindIII/SapI digest, creating pUC19dHS. The digest removedthe lac promoter adjacent to the MCS (multiple cloning site), preventingtranscription of the operons in the vector.

The ald gene was amplified from C. beijerinckii ATCC 35702 genomic DNAby PCR using primers N58 and N59 (see Table 4), given as SEQ ID NOs:41and 42, respectively, creating a 1.5 kbp product. The forward primerincorporated the restriction sites AvaI and BstEII and a RBS (ribosomebinding site). The reverse primer incorporated the HpaI restrictionsite. The PCR product was cloned into pCRBlunt II-TOPO creatingpCRBluntII-ald. Plasmid DNA was prepared from the TOPO clones and thesequence of the genes verified with primers M13 Forward (SEQ ID NO:45),M13 Reverse (SEQ ID NO:46), N31SeqF2 (SEQ ID NO:59), N31SeqF3 (SEQ IDNO:60), N31SeqF4 (SEQ ID NO:61), N32SeqR1 (SEQ ID NO:72), N31SeqR2 (SEQID NO:62), N31SeqR3 SEQ ID NO:63), N31SeqR4 (SEQ ID NO:64), and N31SeqR5(SEQ ID NO:65) (see Table 5).

The bdhB gene was amplified from C. acetobutylicum (ATCC 824) genomicDNA by PCR using primers N64 and N65 (see Table 4), given as SEQ IDNOs:43 and 44, respectively, creating a 1.2 kbp product. The forwardprimer incorporated an HpaI restriction site and a RBS. The reverseprimer incorporated a PmeI and a SphI restriction site. The PCR productwas cloned into pCRBlunt II-TOPO creating pCRBluntII-bdhB. Plasmid DNAwas prepared from the TOPO clones and the sequence of the genes verifiedwith primers M13 Forward (SEQ ID NO:45), M13 Reverse (SEQ ID NO:46),N11SeqF1 (SEQ ID NO:66), N11SeqF2 (SEQ ID NO:67), N12SeqR1 (SEQ IDNO:68), and N12SeqR2 (SEQ ID NO:69) (see Table 5).

To construct the lower pathway operon, a 1.2 kbp SphI and HpaI fragmentfrom pCRBluntII-bdhB, a 1.4 kbp HpaI and SphI fragment frompCRBluntII-ald, and the large fragment from a AvaI and SphI digest ofpUC19dHS were ligated together. The three-way ligation createdpUC19dHS-ald-bdhB.

The pUC19dHS-ald-bdhB vector was digested with BstEII and PmeI releasinga 2.6 kbp fragment that was cloned into pBenBP, an E. coli-Bacillussubtilis shuttle vector. Plasmid pBenBP was created by modification ofthe pBE93 vector, which is described by Nagarajan, WO 93/24631 (Example4). The Bacillus amyloliquefaciens neutral protease promoter (NPR),signal sequence and the phoA gene were removed from pBE93 with aNcoI/HindIII digest. The NPR promoter was PCR amplified from pBE93 byprimers BenF and BenBPR, given by SEQ ID NOs:73 and 75, respectively.Primer BenBPR incorporated BstEII, PmeI and HindIII sites downstream ofthe promoter. The PCR product was digested with NcoI and HindIII and thefragment was cloned into the corresponding sites in the vector pBE93 tocreate pBenBP. The lower operon fragment was subcloned into the BstEIIand PmeI sites in pBenBP creating pBen-ald-bdhB.

Assays for butyraldehyde dehydrogenase and butanol dehydrogenaseactivity were conducted on crude extracts using the methods describedabove.

Both enzyme activities were demonstrated at levels above the controlstrain that contained an empty vector.

Example 14 Prophetic Construction of a Transformation Vector for theGenes in the 1-Butanol Biosynthetic Pathway—Upper Pathway

The purpose of this prophetic Example is to describe how to assemble theupper pathway operon. The general approach is the same as described inExample 13.

The thIA gene is amplified from C. acetobutylicum (ATCC 824) genomic DNAby PCR using primer pair N44 and N45 (see Table 4), given as SEQ IDNOs:33 and 34, respectively, creating a 1.2 kbp product. The forwardprimer incorporates a SphI restriction site and a ribosome binding site(RBS). The reverse primer incorporates AscI and PstI restriction sites.The PCR product is cloned into pCR4 Blunt-TOPO creating pCR4Blunt-TOPO-thIA. Plasmid DNA is prepared from the TOPO clones and thesequence of the genes is verified with primers M13 Forward (SEQ IDNO:45), M13 Reverse (SEQ ID NO:46), N7SeqF1 (SEQ ID NO:47), and N7SeqR1(SEQ ID NO:48) (see Table 5).

The hbd gene is amplified from C. acetobutylicum (ATCC 824) genomic DNAby PCR using primer pair N42 and N43 (see Table 4) given as SEQ IDNOs:35 and 36, respectively, creating a 0.9 kbp product. The forwardprimer incorporates a SalI restriction site and a RBS. The reverseprimer incorporates a SphI restriction site. The PCR product is clonedinto pCR4 Blunt-TOPO creating pCR4 Blunt-TOPO-hbd. Plasmid DNA isprepared from the TOPO clones and the sequence of the genes verifiedwith primers M13 Forward (SEQ ID NO:45), M13 Reverse (SEQ ID NO:46),N5SeqF2 (SEQ ID NO:51), and N6SeqR2 (SEQ ID NO:52) (see Table 5).

The CAC0462 gene is codon optimized for expression in E. coli as primaryhost and B. subtilis as a secondary host. The new gene called CaTER,given as SEQ ID NO:76, is synthesized by Genscript Corp (Piscataway,N.J.). The gene CaTER is cloned in the pUC57 vector as a BamHI-SalIfragment and includes a RBS, producing plasmid pUC57-CaTER.

The crt gene is amplified from C. acetobutylicum (ATCC 824) genomic DNAby PCR using primer pair N38 and N39 (see Table 4), given as SEQ IDNOs:39 and 40, respectively, creating a 834 bp product. The forwardprimer incorporates EcoRI and MluI restriction sites and a RBS. Thereverse primer incorporates a BamHI restriction site. The PCR product iscloned into pCR4 Blunt-TOPO creating pCR4 Blunt-TOPO-crt. Plasmid DNA isprepared from the TOPO clones and the sequence of the genes is verifiedwith primers M13 Forward (SEQ ID NO:45) and M13 Reverse (SEQ ID NO:46)(see Table 5).

After confirmation of the sequence, the genes are subcloned into amodified pUC19 vector as a cloning platform. The pUC19 vector wasmodified by a SphI/SapI digest, creating pUC19dSS. The digest removedthe lac promoter adjacent to the MCS, preventing transcription of theoperons in the vector.

To construct the upper pathway operon pCR4 Blunt-TOPO-crt is digestedwith EcoRI and BamHI releasing a 0.8 kbp crt fragment. The pUC19dSSvector is also digested with EcoRI and BamHI releasing a 2.0 kbp vectorfragment. The crt fragment and the vector fragment are ligated togetherusing T4 DNA ligase (New England Biolabs) to form pUC19dSS-crt. TheCaTER gene is inserted into pCU19dSS-crt by digesting pUC57-CaTER withBamHI and SalI, releasing a 1.2 kbp CaTER fragment. The pUC19dSS-crt isdigested with BamHI and SalI and the large vector fragment is ligatedwith the CaTER fragment, creating pUC19dSS-crt-CaTER. To complete theoperon a 884 bp SalI and SphI fragment from pCR4 Blunt-TOPO-hbd, a 1.2kb SphI and PstI thIA fragment from pCR4 Blunt-TOPO-thIA and the largefragment from a SalI and PstI digest of pUC19dSS-crt-CaTER are ligated.The product of the 3-way ligation is pUC19dSS-crt-CaTER-hbd-thIA.

The pUC19dSS-crt-CaTER-hbd-thIA vector is digested with MluI and AscIreleasing a 4.1 kbp fragment that is cloned into a derivative of pBE93(Caimi, WO2004/018645, pp. 39-40) an E. coli-B. subtilis shuttle vector,referred to as pBenMA. Plasmid pBenMA was created by modification of thepBE93 vector. The Bacillus amyloliquefaciens neutral protease promoter(NPR), signal sequence and the phoA gene are removed from pBE93 with aNcoI/HindIII digest. The NPR promoter is PCR amplified from pBE93 byprimers BenF and BenMAR, given as SEQ ID NOS:73 and 74, respectively.Primer BenMAR incorporates MluI, AscI, and HindIII sites downstream ofthe promoter. The PCR product was digested with NcoI and HindIII and thefragment is cloned into the corresponding sites in the vector pBE93,creating pBenMA. The upper operon fragment is subcloned into the MluIand AscI sites in pBenMA creating pBen-crt-hbd-CaTER-thIA.

Example 15 Prophetic Expression of the 1-Butanol Biosynthetic Pathway inE. coli

The purpose of this prophetic Example is to describe how to express the1-butanol biosynthetic pathway in E. coli.

The plasmids pBen-crt-hbd-CaTER-thIA and pBen-ald-bdhB, constructed asdescribed in Examples 14 and 13, respectively, are transformed into E.coli NM522 (ATCC 47000) and expression of the genes in each operon ismonitored by SDS-PAGE analysis, enzyme assay and Western analysis. ForWesterns, antibodies are raised to synthetic peptides by Sigma-Genosys(The Woodlands, Tex.). After confirmation of expression of all thegenes, pBen-ald-bdhB is digested with EcoRI and PmeI to release the NPRpromoter-ald-bdhB fragment. The EcoRI digest of the fragment is bluntended using the Klenow fragment of DNA polymerase (New England Biolabs,catalog no. M0210S). The plasmid pBen-crt-hbd-CaTER-thIA is digestedwith PvuII to create a linearized blunt ended vector fragment. Thevector and NPR-ald-bdhB fragment are ligated, creating p1B1 O.1 and p1B1O.2, containing the complete 1-butanol biosynthetic pathway with the NPRpromoter-ald-bdhB fragment in opposite orientations. The plasmids p1B1O.1 and p1B1 O.2 are transformed into E. coli NM522 and expression ofthe genes are monitored as previously described.

E. coli strain NM522/p1B1 O.1 or NM522/p1B1 O.1 is inoculated into a 250mL shake flask containing 50 mL of medium and shaken at 250 rpm and 35°C. The medium is composed of: dextrose, 5 g/L; MOPS, 0.05 M; ammoniumsulfate, 0.01 M; potassium phosphate, monobasic, 0.005 M; S10 metal mix,1% (v/v); yeast extract, 0.1% (w/v); casamino acids, 0.1% (w/v);thiamine, 0.1 mg/L; proline, 0.05 mg/L; and biotin 0.002 mg/L, and istitrated to pH 7.0 with KOH. S10 metal mix contains: MgCl₂, 200 mM;CaCl₂, 70 mM; MnCl₂, 5 mM; FeCl₃, 0.1 mM; ZnCl₂, 0.1 mM; thiaminehydrochloride, 0.2 mM; CuSO₄, 172 μM; CoCl₂, 253 μM; and Na₂MoO₄, 242μM. After 18 to 24 h, 1-butanol is detected by HPLC or GC analysis, asdescribed in the General Methods section.

Example 16 Prophetic Expression of the 1-Butanol Biosynthetic Pathway inBacillus subtilis

The purpose of this prophetic Example is to describe how to express the1-butanol biosynthetic pathway in Bacillus subtilis. The same approachas described in Example 15 is used.

The upper and lower operons constructed as described in Examples 14 and13, respectively, are used. The plasmids p1B1 O.1 and p1B1 O.2 aretransformed into Bacillus subtilis BE1010 (J. Bacteriol. 173:2278-2282(1991)) and expression of the genes in each operon is monitored aspreviously described.

B. subtilis strain BE1010/p1B1 O.1 or BE1010/p1B1 O.2 is inoculated intoa 250 mL shake flask containing 50 mL of medium and shaken at 250 rpmand 35° C. for 18 h. The medium is composed of: dextrose, 5 g/L; MOPS,0.05 M; glutamic acid, 0.02 M; ammonium sulfate, 0.01 M; potassiumphosphate, monobasic buffer, 0.005 M; S10 metal mix (as described inExample 15), 1% (v/v); yeast extract, 0.1% (w/v); casamino acids, 0.1%(w/v); tryptophan, 50 mg/L; methionine, 50 mg/L; and lysine, 50 mg/L,and is titrated to pH 7.0 with KOH. After 18 to 24 h, 1-butanol isdetected by HPLC or GC analysis, as described in the General Methodssection.

Example 17 Production of 1-Butanol from Glucose using Recombinant E.coli

This Example describes the production of 1-butanol in E. coli.Expression of the genes encoding the 6 steps of the 1-butanolbiosynthetic pathway was divided into three operons. The upper pathwaycomprised the first four steps encoded by thIA, hbd, crt and EgTER inone operon. The next step, encoded by ald, was provided by a secondoperon. The last step in the pathway, encoded by yqhD, was provided in athird operon. 1-Butanol production was demonstrated in E. coli strainscomprising the three operons.

Unless otherwise indicated in the text, cloning primers described inthis Example are referenced by their SEQ ID NO in Table 4, andsequencing and PCR screening primers are referenced by their SEQ ID NOin Table 5.

Acetyl-CoA Acetyltransferase.

The thIA gene was amplified from C. acetobutylicum (ATCC 824) genomicDNA by PCR using primer pair N44 and N45 (see Table 4), given as SEQ IDNOs:33 and 34, respectively, creating a 1.2 kbp product. The forwardprimer incorporated a SphI restriction site and a ribosome binding site(RBS). The reverse primer incorporated AscI and PstI restriction sites.The PCR product was cloned into pCR4Blunt-TOPO (Invitrogen Corp.,Carlsbad, Calif.) creating pCR4Blunt-TOPO-thIA. Plasmid DNA was preparedfrom the TOPO clones and the sequence of the genes was verified withprimers M13 Forward (SEQ ID NO:45), M13 Reverse (SEQ ID NO:46), N7SeqF1(SEQ ID NO:47), and N7SeqR1 (SEQ ID NO:48) (see Table 5).

3-Hydroxybutyryl-CoA dehydrogenase.

The hbd gene was amplified from C. acetobutylicum (ATCC 824) genomic DNAby PCR using primer pair N42 and N43 (see Table 4) given as SEQ IDNOs:35 and 36, respectively, creating a 0.9 kbp product. The forwardprimer incorporated a SalI restriction site and a RBS. The reverseprimer incorporated a SphI restriction site. The PCR product was clonedinto pCR4Blunt-TOPO creating pCR4Blunt-TOPO-hbd. Plasmid DNA wasprepared from the TOPO clones and the sequence of the genes verifiedwith primers M13 Forward (SEQ ID NO:45), M13 Reverse (SEQ ID NO:46),N5SeqF2 (SEQ ID NO:51), and N6SeqR2 (SEQ ID NO:52) (see Table 5).

Crotonase.

The crt gene was amplified from C. acetobutylicum (ATCC 824) genomic DNAby PCR using primer pair N38 and N39 (see Table 4), given as SEQ IDNOs:39 and 40, respectively, creating a 834 bp product. The forwardprimer incorporated EcoRI and MluI restriction sites and a RBS. Thereverse primer incorporated a BamHI restriction site. The PCR productwas cloned into pCR4Blunt-TOPO creating pCR4Blunt-TOPO-crt. Plasmid DNAwas prepared from the TOPO clones and the sequence of the genes wasverified with primers M13 Forward (SEQ ID NO:45) and M13 Reverse (SEQ IDNO:46) (see Table 5).

Butyryl-CoA Dehydrogenase (trans-2-enoyl-CoA reductase).

The CAC0462 gene was synthesized for enhanced codon usage in E. coli asprimary host and B. subtilis as a secondary host. The new gene (CaTER,SEQ ID NO:76) was synthesized and cloned by Genscript Corporation(Piscataway, N.J.) in the pUC57 vector as a BamHI-SalI fragment andincludes a RBS.

An alternative gene for butyryl-CoA dehydrogenase from Euglena gracilis(TER, GenBank No. Q5EU90) was synthesized for enhanced codon usage in E.coli and Bacillus subtilis. The gene was synthesized and cloned byGenScript Corporation into pUC57 creating pUC57::EgTER. Primers N85 andN86, (SED ID NO: 80 and 81 respectively) together with pUC57::EgTER astemplate DNA, provided a PCR fragment comprising 1224 bp frompUC57::EgTER DNA. The sequence of the 1224 bp is given as SEQ ID NO:77,where bp 1-1218 is the coding sequence (cds) of EgTER(opt). EgTER(opt)is a codon optimized TER gene, lacking the normal mitochondrialpresequence so as to be functional in E. coli (Hoffmeister et al., J.Biol. Chem. 280:4329 (2005)).

EgTER(opt) was cloned into pCR4Blunt-TOPO and its sequence was confirmedwith primers M13 Forward (SEQ ID NO:45) and M13 Reverse (SEQ ID NO:46).Additional sequencing primers N62SeqF2 (SEQ ID NO:114), N62SeqF3 (SEQ IDNO:115), N62SeqF4 (SEQ ID NO:116), N63SeqR1 (SEQ ID NO:117), N63SeqR2(SEQ ID NO:118), N63SeqR3 (SEQ ID NO:119) and N63SeqR4 (SEQ ID NO:120)were needed to completely sequence the PCR product. The 1.2 kbpEgTER(opt) sequence was then excised with HincII and PmeI and clonedinto pET23+ (Novagen) linearized with HincII. Orientation of theEgTER(opt) gene to the promoter was confirmed by colony PCR screeningwith primers T7Primer and N63SeqR2 (SEQ ID NOs:82 and 118 respectively).The resulting plasmid, pET23+::EgTER(opt), was transformed into BL21(DE3) (Novagen) for expression studies.

Trans-2-enoyl-CoA reductase activity was assayed as described byHoffmeister et al., J. Biol. Chem. 280:4329 (2005). In a typical assay,the specific activity of the EgTER(opt) protein in the induced BL21(DE3) /pET23+::EgTER(opt) culture was determined to be 1.9 μmol mg⁻¹min⁻¹ compared to 0.547 μmol mg⁻¹ min⁻¹ in the uninduced culture.

The EgTER(opt) gene was then cloned into the pTrc99a vector under thecontrol of the trc promoter. The EgTER(opt) gene was isolated as a1287-bp BamHI/SalI fragment from pET23+::EgTER(opt). The 4.2 kbp vectorpTrc99a was linearized with BamHI/SalI. The vector and fragment wereligated creating the 5.4 kbp pTrc99a-EgTER(opt). Positive clones wereconfirmed by colony PCR with primers Trc99aF and N63SeqR3 (SEQ ID NOs:83and 119 respectively) producing a 0.5 kb product.

Construction of Plasmid pTrc99a-E-C-H-T Comprising Genes EncodingAcetyl-CoA Acetyltransferase (thIA), 3-Hydroxybutyryl-CoA Dehydrogenase(hbd), Crotonase (Crt), and Butyryl-CoA Dehydrogenase (Trans-2-Enoyl-CoAReductase, EgTER(opt)).

To initiate the construction of a four gene operon comprising the upperpathway (EgTER(opt), crt, hbd and thIA), pCR4Blunt-TOPO-crt was digestedwith EcoRI and BamHI releasing a 0.8 kbp crt fragment. The pUC19dSSvector (described in Example 14) was also digested with EcoRI and BamHIreleasing a 2.0 kbp vector fragment. The crt fragment and the vectorfragment were ligated together using T4 DNA ligase (New England Biolabs)to form pUC19dSS-crt. The CaTER gene was inserted into pUC19dSS-crt bydigesting pUC57-CaTER with BamHI and SalI, releasing a 1.2 kbp CaTERfragment. The pUC19dSS-crt was digested with BamHI and SalI and thelarge vector fragment was ligated with the CaTER fragment, creatingpUC19dSS-crt-CaTER. To complete the operon a 884 bp SalI and SphIfragment from pCR4Blunt-TOPO-hbd, a 1.2 kb SphI and PstI thIA fragmentfrom pCR4Blunt-TOPO-thIA and the large fragment from a SalI and PstIdigest of pUC19dSS-crt-CaTER were ligated. The product of the 3-wayligation was named pUC19dSS-crt-CaTER-hbd-thIA or pUC19dss::Operon1.

Higher butyryl-CoA dehydrogenase activity was obtained frompTrc99a-EgTER(opt) than from CaTER constructs, thus, an operon derivedfrom pTrc99a-EgTER(opt) was constructed. The CaTER gene was removed frompUC19dss::Operon1 by digesting with BamHI/Sal I and gel purifying the5327-bp vector fragment. The vector was treated with Klenow andreligated creating pUC19dss::Operon 1 ΔCaTer. The 2934-bp crt-hbd-thIA(C-H-T) fragment was then isolated as a EcoRI/PstI fragment frompUC19dss:Operon 1 ACaTer. The C-H-T fragment was treated with Klenow toblunt the ends. The vector pTrc99a-EgTER(opt) was digested with SalI andthe ends treated with Klenow. The blunt-ended vector and the blunt-endedC-H-T fragment were ligated to create pTrc99a-E-C-H-T. Colony PCRreactions were performed with primers N62SeqF4 and N5SeqF4 (SEQ ID NOs:116 and 84 respectively) to confirm the orientation of the insert.

Construction of Plasmids pBHR T7-ald and pBHR-Ptrc-ald(Opt) ComprisingGenes Encoding Butyraldehyde Dehydrogenase (ald and ald(opt)).

The PT7-ald operon was sub-cloned from pDEST14ald (Example 10) into thebroad host range plasmid pBHR1 (MoBitec, Goettingen, Germany) to createpBHR1 PT7-ald. The pBHRi plasmid is compatible with pUC19 or pBR322plasmids so pBHR1 PT7-ald can be used in combination with pUC19 orpBR322 derivatives carrying the upper pathway operon for 1-butanolproduction in E. coli. The pDEST14-ald plasmid was digested with Bgl IIand treated with the Klenow fragment of DNA polymerase to make bluntends. The plasmid was then digested with EcoRI and the 2,245 bp PT7-aldfragment was gel-purified. Plasmid pBHR1 was digested with ScaI andEcoRI and the 4,883 bp fragment was gel-purified. The PT7-ald fragmentwas ligated with the pBHR1 vector, creating pBHR T7-ald. Colony PCRamplification of transformants with primers T-ald(BamHI) andB-ald(EgTER) (SEQ ID NOs:85 and 86 respectively) confirmed the expected1.4 kb PCR product. Restriction mapping of pBHR T7-ald clones with EcoRIand DrdI confirmed the expected 4,757 and 2,405 bp fragments.

For butyraldehyde dehydrogenase activity assays, the plasmid pBHR T7-aldwas transformed into BL21Star™ (DE3) cells (Invitrogen) and expressionfrom the T7 promoter was induced by addition of L-arabinose as describedin Example 5. Acylating aldehyde dehydrogenase activity was determinedby monitoring the formation of NADH, as measured by the increase inabsorbance at 340 nm, as described in Example 10.

An alternative DNA sequence for the ald gene from Clostridiumbeijerinckii ATCC 35702 was synthesized (optimizing for codon usage inE. coli and Bacillus subtilis) and cloned into pUC57 bp GenScriptCorporation (Piscataway, N.J.), creating the plasmid pUC57-ald(opt).pUC57-ald(opt) was digested with SacI and SalI to release a 1498 bpfragment comprising the condon optimized gene, ald(opt) and a RBSalready for E. coli. The sequence of the 1498 bp fragment is given asSEQ ID NO:78.

pTrc99a was digested with SacI and Sail giving a 4153 bp vectorfragment, which was ligated with the 1498 bp ald(opt) fragment to createpTrc-ald(opt). Expression of the synthetic gene, ald(opt), is under thecontrol of the IPTG-inducible Ptrc promoter.

The Ptrc-ald(opt) operon was subcloned into the broad host range plasmidpBHR1 (MoBitec) in order to be compatible with the upper pathway plasmiddescribed above. The Ptrc-ald(opt) fragment was PCR-amplified frompTrc99A::ald(opt) with T-Ptrc(BspEI) and B-aldopt(ScaI), (SEQ ID NOs:87and 88 respectively) incorporating BspEI and ScaI restriction siteswithin the corresponding primers. The PCR product was digested withBspEI and ScaI. The plasmid pBHR1 was digested with ScaI and BspEI andthe 4,883 bp fragment was gel-purified. The Ptrc-ald(opt) fragment wasligated with the pBHR1 vector, creating pBHR-PcatPtrc-ald(opt).Restriction mapping of the pBHR-PcatPtrc-ald(opt) clones with ScaI andBspEI confirmed the expected 4,883 and 1,704 bp fragments. To remove theplasmid-born cat promoter (Pcat) region, plasmid pBHR-PcatPtrc-ald(opt)was digested with BspEI and AatII and the 6,172 bp fragment wasgel-purified. T-BspEIAatII and B-BspEIAatII (SEQ ID NOs:89 and 90respectively) were mixed in a solution containing 50 mM NaCl, 10 mMTris-HCl, and 10 mM MgCl₂ (pH7.9) to a final concentration of 100 μM andhybridized by incubating at 75° C. for 5 min and slowly cooling to roomtemperature. The hybridized oligonucleotides were ligated with the 6,172bp fragment, creating pBHR-Ptrc-ald(opt).

Construction of E. Coli Strains Expressing Butanol Dehydrogenase (yghD).

E. coli contains a native gene (yqhD) that was identified as a1,3-propanediol dehydrogenase (U.S. Pat. No. 6,514,733). The yqhD genehas 40% identity to the gene adhB in Clostridium, a probableNADH-dependent butanol dehydrogenase. The yqhD gene was placed under theconstitutive expression of a variant of the glucose isomerase promoter1.6GI (SEQ ID NO:91) in E. coli strain MG1655 1.6yqhD::Cm (WO2004/033646) using Red technology (Datsenko and Wanner, Proc. Natl.Acad. Sci. U.S.A. 97:6640 (2000)). Similarly, the native promoter wasreplaced by the 1.5GI promoter (WO 2003/089621) (SEQ ID NO:92), creatingstrain MG1655 1.5GI-yqhD::Cm, thus, replacing the 1.6GI promoter ofMG1655 1.6yqhD::Cm with the 1.5GI promoter.

A P1 lysate was prepared from MG1655 1.5GI yqhD::Cm and the cassettemoved to expression strains, MG1655 (DE3), prepared from E. coli strainMG1655 and a lambda DE3 lysogenization kit (Invitrogen), and BL21 (DE3)(Invitrogen) creating MG1655 (DE3) 1.5GI-yqhD::Cm and BL21 (DE3)1.5GI-yqhD::Cm, respectively.

Demonstration Of 1-Butanol Production from Recombinant E. Coli.

E. coli strain MG1655 (DE3) 1.5GI-yqhD::Cm was transformed with plasmidspTrc99a-E-C-H-T and pBHR T7-ald to produce the strain, MG1655 (DE3)1.5GI -yqhD::Cm/pTrc99a-E-C-H-T/pBHR T7-ald. Two independent isolateswere initially grown in LB medium containing 50 μg/mL kanamycin and 100μg/mL carbenicillin. The cells were used to inoculate shake flasks(approximately 175 mL total volume) containing 15, 50 and 150 mL ofTM3a/glucose medium (with appropriate antibiotics) to represent high,medium and low oxygen conditions, respectively. TM3a/glucose mediumcontained (per liter): 10 g glucose, 13.6 g KH₂PO₄, 2.0 g citric acidmonohydrate, 3.0 g (NH₄)₂SO₄, 2.0 g MgSO₄.7H₂O, 0.2 g CaCl₂2H₂O, 0.33 gferric ammonium citrate, 1.0 mg thiamine.HCl, 0.50 g yeast extract, and10 mL trace elements solution, adjusted to pH 6.8 with NH₄OH. Thesolution of trace elements contained: citric acid .H₂O (4.0 g/L),MnSO₄.H₂O (3.0 g/L), NaCl (1.0 g/L), FeSO₄.7H₂O (0.10 g/L), CoCl₂.6H₂O(0.10 g/L), ZnSO₄. 7H₂O (0.10 g/L), CuSO₄.5H₂O (0.010 g/L), H₃BO₃ (0.010g/L), and Na₂MoO₄. 2H₂O (0.010 g/L). The flasks were inoculated at astarting OD₆₀₀ of 0.01 units and incubated at 34° C. with shaking at 300rpm. The flasks containing 15 and 50 mL of medium were capped withvented caps; the flasks containing 150 mL, were capped with non-ventedcaps to minimize air exchange. IPTG was added to a final concentrationof 0.04 mM; the OD₆₀₀ of the flasks at the time of addition was ≧0.4units.

Approximately 15 h after induction, an aliquot of the broth was analyzedby HPLC (Shodex Sugar SH1011 column) with refractive index (R1)detection and GC (Varian CP-WAX 58(FFAP) CB column, 25 m×0.25 mm id×0.2μm film thickness) with flame ionization detection (FID) for 1-butanolcontent, as described in the General Methods section.

The results of the 1-butanol determinations are given in Table 14.

TABLE 14 Production of 1-butanol by E. coli strain MG1655 (DE3)1.5GI-yqhD::Cm/pTrc99a-E-C-H-T/pBHR T7-ald. Strain O₂ Level 1-butanol,mM molar yield, % MG1655 a high 0.11 0.2 MG1655 b high 0.12 0.2 MG1655 amedium 0.13 0.3 MG1655 b medium 0.13 0.2 MG1655 a low 0.15 0.4 MG1655 blow 0.18 0.5 Values were determined from HPLC analysis. Strain suffixes“a” and “b” indicate independent isolates.

The two independent isolates of MG1655 (DE3)1.5GI-yqhD::Cm/pTrc99a-E-C-H-T/pBHR T7-ald were tested for 1-butanolproduction in an identical manner except that the medium contained 5 g/Lyeast extract. The results are shown in Table 15.

TABLE 15 Production of 1-butanol by E. coli strain MG1655 (DE3)1.5GI-yqhD::Cm/pTrc99a-E-C-H-T/pBHR T7-ald. Strain O₂ Level 1-butanol,mM molar yield, % MG1655 a high − − MG1655 b high − − MG1655 a medium0.08 0.1 MG1655 b medium 0.06 0.1 MG1655 a low 0.14 0.3 MG1655 b low0.14 0.3 Quantitative values were determined from HPLC analysis. “−” =not detected. Strain suffixes “a” and “b” indicate independent isolates.

E. coli strain BL21 (DE3) 1.5GI-yqhD::Cm was transformed with plasmidspTrc99a-E-C-H-T and pBHR T7-ald to produce the strain, BL21 (DE3) 1.5GI-yqhD::Cm/pTrc99a-E-C-H-T/pBHR T7-ald. Two independent isolates weretested for 1-butanol production exactly as described above. The resultsare given in Tables 16 and 17.

TABLE 16 Production of 1-butanol by E. coli strain BL21 (DE3)1.5GI-yqhD::Cm/pTrc99a-E-C-H-T/pBHR T7-ald. Strain O₂ Level 1-butanol,mM molar yield, % DE a high + + DE b high − − DE a medium 0.80 1.4 DE bmedium 0.77 1.4 DE a low 0.06 0.2 DE b low 0.07 0.2 Quantitative valueswere determined from HPLC analysis. “−” indicates not detected. “+”indicates positive, qualitative identification by GC with a lowerdetection limit than with HPLC. Strain suffixes “a” and “b” indicateindependent isolates.

TABLE 17 Production of 1-butanol by E. coli strain BL21 (DE3)1.5GI-yqhD::Cm/pTrc99a-E-C-H-T/pBHR T7-ald. Strain O₂ Level 1-butanol,mM molar yield, % DE a high + + DE b high + + DE a medium 0.92 1.7 DE bmedium 1.03 1.9 DE a low + + DE b low + + Quantitative values weredetermined from HPLC analysis. “−” indicates not detected. “+” indicatespositive, qualitative identification by GC with a lower detection limitthan with HPLC. Strain suffixes “a” and “b” indicate independentisolates.

E. coli strain MG1655 1.5GI-yqhD::Cm was transformed with plasmidspTrc99a-E-C-H-T and pBHR-Ptrc-ald(opt) to produce the strain, MG16551.5GI -yqhD::Cm/pTrc99a-E-C-H-T/pBHR-Ptrc-ald(opt). Two isolates wereinitially grown in LB medium containing 50 μg/mL kanamycin and 100 μg/mLcarbenicillin. The cells were used to inoculate shake flasks(approximately 175 mL total volume) containing 50 and 150 mL ofTM3a/glucose medium (with appropriate antibiotics). The flasks wereinoculated at a starting OD₅₅₀ of 0.04 units and incubated as describedabove, with and without induction. IPTG was added to a finalconcentration of 0.4 mM; the OD₅₅₀ of the flasks at the time of additionwas between 0.6 and 1.2 units. In this case, induction was not necessaryfor 1-butanol pathway gene expression because of the leakiness of theIPTG inducible promoters and the constitutive nature of the 1.5GIpromoter; however, induction provided a wider range of expression.

Approximately 15 h after induction, an aliquot of the broth was analyzedby GC with flame ionization detection for 1-butanol content, asdescribed above. The results are given in Table 18. For the recombinantE. coli strains, 1-butanol was produced in all cases; in separateexperiments, wild type E. coli strains were shown to produce nodetectable 1-butanol (data not shown).

TABLE 18 Production of 1-butanol by E. coli strain MG16551.5GI-yqhD::Cm/pTrc99a-E-C-H-T/pBHR-Ptrc-ald(opt). Strain O₂ Level1-butanol, mM IPTG Induction MG1655 a medium 0.14 No MG 1655 b medium0.14 No MG1655 a medium 0.03 Yes MG 1655 b medium 0.07 Yes MG1655 a low0.04 No MG 1655 b low 0.04 No MG1655 a low 0.02 Yes MG 1655 b low 0.03Yes Strain suffixes “a” and “b” indicate separate isolates.

Example 18 Production of 1-Butanol from Glucose Using Recombinant B.Subtilis

This Example describes the production of 1-butanol in Bacillus subtilis.The six genes of the 1-biosynthetic pathway, encoding six enzymeactivities, were split into two operons for expression. The first threegenes of the pathway (thI, hbd, and crt) were integrated into thechromosome of Bacillus subtilis BE1010 (Payne and Jackson, J. Bacteriol.173:2278-2282 (1991)). The last three genes (EgTER, ald, and bdhB) werecloned into an expression plasmid and transformed into the Bacillusstrain carrying the integrated 1-butanol genes.

Unless otherwise indicated in the text, cloning primers described inthis Example are referenced by their SEQ ID NO in Table 4, andsequencing and PCR screening primers are referenced by their SEQ ID NOin Table 5.

Integration Plasmid.

Plasmid pFP988 is a Bacillus integration vector that contains an E. colireplicon from pBR322, an ampicillin antibiotic marker for selection inE. coli and two sections of homology to the sacB gene in the Bacilluschromosome that directs integration of the vector and interveningsequence by homologous recombination. Between the sacB homology regionsis the Pamy promoter and signal sequence that can direct the synthesisand secretion of a cloned gene, a His-Tag and erythromycin as aselectable marker for Bacillus. The Pamy promoter and signal sequence isfrom Bacillus amyloliquefaciens alpha-amylase. The promoter region alsocontains the lacO sequence for regulation of expression by a lacIrepressor protein. The sequence of pFP988 (6509 bp) is given as SEQ IDNO:79.

Since the 1-butanol pathway genes were to be expressed in the cytoplasm,the amylase signal sequence was deleted. Plasmid pFP988 was amplifiedwith primers Pamy/lacO F and Pamy/lacO R creating a 317 bp (0.3 kbp)product that contained the Pamy/lacO promoter. The 5′ end of thePamy/lacO F primer incorporated a BsrGI restriction site followed by anEcoRI site. The 5′ end of the Pamy/lacO R primer incorporated a BsrGIrestriction site followed by a PmeI restriction site. The PCR productwas TOPO cloned into pCR4Blunt-TOPO creating pCR4Blunt-TOPO-Pamy/lacO.Plasmid DNA was prepared from overnight cultures and submitted forsequencing with M13 Forward and M13 Reverse primers (SEQ ID NO:45 andSEQ ID NO:46, respectively) to ensure no mutation had been introducedinto the promoter. A clone of pCR4Blunt-TOPO-Pamy/lacO was digested withBsrGI and the 0.3 kbp fragment was gel-purified. The vector pFP988 wasdigested with BsrGI resulting in deletion of 11 bp from the 5′ sacBhomology region and the removal of the Pamy/lacO promoter and signalsequence and His-tag. The 6 kbp BsrGI digested vector was gel-purifiedand ligated with Pamy/lacO BsrGI insert. The resulting plasmids werescreened with primers Pamy SeqF2 and Pamy SeqR to determine orientationof the promoter. The correct clone restored the Pamy/lacO promoter toits original orientation and was named pFP988Dss.

The cassette with genes thI-crt was constructed by SOE (splicing byoverlap extension). The genes were amplified using as templatepUC19dss::Operon1. The thI primers were Top TF and Bot TR amplifying a0.9 kbp product. The crt primers were Top CF and Bot CR amplifying a 1.3kbp product. The two genes were joined by SOE with PCR amplificationusing primers Top TF and Bot CR generating a 2.1 kbp product that wasTOPO cloned into pCR4Blunt-TOPO creating pCR4Blunt-TOPO-T-C. Clones weresubmitted for sequencing to confirm the sequence. The plasmidpCR4Blunt-TOPO-T-C was digested with BstEII and PmeI releasing a 2.1 kbpfragment that was gel-purified. The insert was treated with Klenowpolymerase to blunt the BstEII site. Vector pFP988Dss was digested withPmeI and treated with calf intestinal alkaline phosphatase (New EnglandBioLabs) to prevent self-ligation. The 2.1 kbp thI-crt fragment and thedigested pFP988Dss were ligated and transformed into E. coli Top10cells. Transformants were screened by PCR amplification with Pamy SeqF2and N7SeqR2 for a 0.7 kbp product, the correct product was calledpFP988Dss-T-C.

Construction of the thI-crt cassette created unique SalI and SpeI sitesbetween the two genes. To add the hbd gene to the cassette, the hbd genewas subcloned from pCR4Blunt-TOPO-hbd as a 0.9 kbp SalI/SpeI fragment.Vector pFP988Dss-T-C was digested with SalI and SpeI and the 8 kbpvector fragment was gel-purified. The vector and hbd insert were ligatedand transformed into E. coli Top10 cells. Transformants were screened byPCR amplification with primers Pamy SeqF and N3SeqF3 for a 3.0 kbpfragment. The resulting plasmid was named pFP988Dss-T-H-C.

The Pamy promoter subsequently was replaced with the Pspac promoter fromplasmid pMUTIN4 (Vagner et al., Microbiol. 144:3097-3104 (1998)). ThePspac promoter was amplified from pMUTIN4 with primers Spac F and Spac Ras a 0.4 kbp product and TOPO cloned into pCR4Blunt-TOPO. Transformantswere screened by PCR amplification with M13 Forward and M13 Reverseprimers for the presence of a 0.5 kbp insert. Positive clones weresubmitted for sequencing with the same primers. PlasmidpCR4Blunt-TOPO-Pspac was digested with SmaI and XhoI and the 0.3 kbpfragment was gel-purified. Vector pFP988Dss-T-H-C was digested with SmaIand XhoI and the 9 kbp vector was isolated by gel purification. Thedigested vector and Pspac insert were ligated and transformed into E.coli Top10 cells. Transformants were screened by PCR amplification withprimers SpacF Seq and N7SeqR2. Positive clones gave a 0.7 kbp product.Plasmid DNA was prepared from positive clones and further screened byPCR amplification with primers SpacF Seq and N3SeqF2. Positive clonesgave a 3 kbp PCR product and were named pFP988DssPspac-T-H-C.

Integration into B. subtilis BE1010 to Form B. subtilisΔsacB::T-H-C::erm #28 Comprising Exogenous thI, hbd, and crt Genes.

Competent cells of B. subtilis BE1010 were prepared as described inDoyle et al., J. Bacteriol. 144:957-966 (1980). Competent cells wereharvested by centrifugation and the cell pellets were resuspended in asmall volume of the cell supernatant. To 1 volume of competent cells, 2volumes of SPII-EGTA medium (Methods for General and MolecularBacteriology, P. Gerhardt et al., Eds, American Society forMicrobiology, Washington, D.C. (1994)) was added. Aliquots of 0.3 mL ofcells were dispensed into test tubes and the plasmidpFP988DssPspac-T-H-C was added to the tubes. Cells were incubated for 30minutes at 37° C. with shaking, after which 0.1 mL of 10% yeast extractwas added to each tube and the cells were further incubated for 60 min.Transformants were plated for selection on LB erythromycin plates usingthe double agar overlay method (Methods for General and MolecularBacteriology, supra). Transformants were initially screened by PCRamplification with primers Pamy SeqF and N5SeqF3. Positive clones thatamplified the expected 2 kbp PCR product were further screened by PCRamplification. If insertion of the cassette into the chromosome hadoccurred via a double crossover event then primer set sacB Up andN7SeqR2 and primer set sacB Dn and N4SeqR3 would amplify a 1.7 kbp and a2.7 kbp product respectively. A positive clone was identified and namedB. subtilis ΔsacB::T-H-C::erm #28.

Plasmid Expression of EgTER, ald, and bdhB Genes.

The three remaining 1-butanol genes were expressed from plasmid pHT01(MoBitec). Plasmid pHT01 is a Bacillus-E. coli shuttle vector thatreplicates via a theta mechanism. Cloned proteins are expressed from theGroEL promoter fused to a lacO sequence. Downstream of the lacO is theefficient RBS from the gsiB gene followed by a MCS. The ald gene wasamplified by PCR with primers AF BamHI and AR Aat2 usingpUC19dHS-ald-bdhB (described in Example 13) as template, creating a 1.4kbp product. The product was TOPO cloned into pCR4-TOPO and transformedinto E. coli Top10 cells. Transformants were screened with M13 Forwardand M13 Reverse primers. Positive clones amplified a 1.6 kbp product.Clones were submitted for sequencing with primers M13 forward and M13reverse, N31SeqF2, N31SeqF3, N32SeqR2, N32SeqR3 and N32SeqR4. Theplasmid was named pCR4TOPO-B/A-ald.

Vector pHT01 and plasmid pCR4TOPO-B/A-ald were both digested with BamHIand AatII. The 7.9 kbp vector fragment and the 1.4 kbp ald fragment wereligated together to create pHT01-ald. The ligation was transformed intoE. coli Top10 cells and transformants were screened by PCR amplificationwith primers N31SeqF1 and HT R for a 1.3 kbp product. To add the lasttwo steps of the pathway to the pHT01 vector, two cloning schemes weredesigned. For both schemes, EgTER and bdhB were amplified together bySOE. Subsequently, the EgTER-bdh fragment was either cloned intopHT01-ald creating pHT01-ald-EB or cloned into pCR4-TOPO-B/A-aldcreating pCR4-TOPO-ald-EB. The ald-EgTer-bdhB fragment from the TOPOvector was then cloned into pHT01 creating pHT01-AEB.

An EgTER-bdhB fragment was PCR amplified using primers Forward 1 (E) andReverse 2 (B), using template DNA given as SEQ ID NO:208. The resulting2.5 kbp PCR product was TOPO cloned into pCR4Blunt-TOPO, creatingpCR4Blunt-TOPO-E-B. The TOPO reaction was transformed into E. coli Top10cells. Colonies were screened with M13 Forward and M13 Reverse primersby PCR amplification. Positive clones generated a 2.6 kbp product.Clones of pCR4Blunt-TOPO-E-B were submitted for sequencing with primersM13 Forward and Reverse, N62SeqF2, N62SeqF3, N62SeqF4, N63SeqR1,N63SeqR2, N63SeqR3, N11Seq F1 and N11Seq F2, N12SeqR1 and N12SeqR2.

Plasmid pCR4Blunt-TOPO-E-B was digested with HpaI and AatII to release a2.4 kbp fragment. The E-B fragment was treated with Klenow polymerase toblunt the end and then was gel-purified. Plasmid pHT01-ald was digestedwith AatII and treated with Klenow polymerase to blunt the ends. Thevector was then treated with calf intestinal alkaline phosphatase andwas gel-purified. The E-B fragment was ligated to the linearized vectorpHT01-ald, transformed into E. coli Top10 cells, and selected on LBplates containing 100 μg/mL ampicillin. Transformants were screened byPCR amplification with primers N3SeqF1 and N63SeqR1 to give a 2.4 kbpproduct. The resulting plasmid, pHT01-ald-EB, was transformed into JM103cells, a recA⁺ E. coli strain. Plasmids prepared from recA⁺ strains formmore multimers than recA⁻ strains. Bacillus subtilis transforms moreefficiently with plasmid multimers rather than monomers (Methods forGeneral and Molecular Bacteriology, supra). Plasmid DNA was preparedfrom JM103 and transformed into competent B. subtilis ΔsacB::T-H-C::erm#28 forming strain B. subtilis ΔsacB::T-H-C::erm #28/pHT01-ald-EB.Competent cells were prepared and transformed as previously described.Transformants were selected on LB plates containing 5 μg/mLchloramphenicol and screened by colony PCR with the primers N31SeqF1 andN63SeqR4 for a 1.3 kbp product.

In the alternate cloning strategy, pCR4Blunt-TOPO-E-B was digested withHpaI and AatII releasing a 2.4 kbp fragment that was gel-purified.Plasmid pCR4-TOPO-B/A-ald was digested with HpaI and AatII and the 5.4kbp vector fragment was gel-purified. The vector fragment frompCR4-TOPO-B/A-ald was ligated with the HpaI-AatII E-B fragment creatingpCR4-TOPO-ald-EB. The ligation was transformed into E. coli Top10 cellsand the resulting transformants were screened by PCR amplification withprimers N11SeqF2 and N63SeqR4 for a 2.1 kbp product. PlasmidpCR4-TOPO-ald-EB was digested with BamHI and AatII and SphI. TheBamHI/AatII digest releases a 3.9 kbp ald-EB fragment that wasgel-purified. The purpose of the SphI digest was to cut the remainingvector into smaller fragments so that it would not co-migrate on a gelwith the ald-EB insert. Vector pHT01 was digested with BamHI and AatIIand the 7.9 kbp vector fragment was gel-purified. The vector and ald-EBinsert fragments were ligated to form plasmid pHT01-AEB and transformedinto E. coli Top10 cells. Colonies were screened by PCR amplificationwith primers N62SeqF4 and HT R for a 1.5 kbp product. Plasmid wasprepared and transformed into JM103. Plasmid DNA was prepared from JM103and transformed into competent B. subtilis ΔsacB::T-H-C::erm #28 formingstrain B. subtilis ΔsacB::T-H-C::erm #28/pHT01-AEB. Competent BE1010cells were prepared and transformed as previously described. Bacillustransformants were screened by PCR amplification with primers N31SeqF1and N63SeqR4 for a 1.3 kbp product.

Demonstration of 1-Butanol Production from Recombinant B. Subtilis.

Three independent isolates of each strain of B. subtilisΔsacB::T-H-C::erm #28/pHT01-ald-EB and B. subtilis ΔsacB::T-H-C::erm#28/pHT01-AEB were inoculated into shake flasks (approximately 175 mLtotal volume) containing 15 mL of medium. A B. subtilis BE1010 strainlacking the exogenous 1-butanol, six gene pathway was also included as anegative control. The medium contained (per liter): 10 mL of 1 M(NH₄)₂SO₄; 5 mL of 1 M potassium phosphate buffer, pH 7.0; 100 mL of 1 MMOPS/KOH buffer, pH 7.0; 20 mL of 1 M L-glutamic acid, potassium salt;10 g glucose; 10 mL of 5 g/L each of L-methionine, L-tryptophan, andL-lysine; 0.1 g each of yeast extract and casamino acids; 20 mL of metalmix; and appropriate antibiotics (5 mg chloramphenicol and erythromycinfor the recombinant strains). The metal mix contained 200 mM MgCl₂, 70mM CaCl₂, 5 mM MnCl₂, 0.1 mM FeCl₃, 0.1 mM ZnCl₂, 0.2 mM thiaminehydrochloride, 172 μM CuSO₄, 253 μM CoCl₂, and 242 μM Na₂MoO₄. Theflasks were inoculated at a starting OD₆₀₀ of 0.1 units, sealed withnon-vented caps, and incubated at 37° C. with shaking at approximately200 rpm.

Approximately 24 h after inoculation, an aliquot of the broth wasanalyzed by HPLC (Shodex Sugar SH1011 column) with refractive index (R1)detection and GC (Varian CP-WAX 58(FFAP) CB column, 0.25 mm×0.2 μm×25 m)with flame ionization detection (FID) for 1-butanol content, asdescribed in the General Methods section. The results of the 1-butanoldeterminations are given in Table 19.

TABLE 19 Production of 1-butanol by strains B. subtilisΔsacB::T-H-C::erm #28/pHT01-ald-EB and B. subtilis ΔsacB::T-H-C::erm#28/pHT01-AEB Strain 1-butanol, HPLC RI peak area 1-butanol, mM* BE1010control Not detected Not detected pHT01-ald-EB a 4629 0.19 pHT01-ald-EBb 3969 Not determined pHT01-ald-EB c 4306 Not determined pHT01-AEB a4926 0.16 pHT01-AEB b 3984 Not determined pHT01-AEB c 3970 Notdetermined *Concentration determined by GC. Strain suffixes “a”, “b”,and “c” indicate separate isolates.

Example 19 Production of 1-Butanol from Glucose or Sucrose byRecombinant E. Coli

To endow E. coli MG1655 with the ability to use sucrose as the carbonand energy source for 1-butanol production, a sucrose utilization genecluster (cscBKA) from plasmid pScrI (described below) was subcloned intopBHR-Ptrc-ald(opt) (described in Example 17) in this organism. Thesucrose utilization genes (cscA, cscK, and cscB) encode a sucrosehydrolase (CscA), given as SEQ ID NO:157, D-fructokinase (CscK), givenas SEQ ID NO:158, and sucrose permease (CscB), given as SEQ ID NO:159.To allow constitutive expression of the three genes from their naturalpromoter, the sucrose-specific repressor gene, cscR, that regulates thegene cluster is not present in the construct.

Cloning and Expression of the Sucrose Utilization Gene Cluster cscBKA inPlasmid pBHR-Ptrc-ald(opt).

The sucrose utilization gene cluster cscBKA, given as SEQ ID NO:156, wasisolated from genomic DNA of a sucrose-utilizing E. coli strain derivedfrom E. coli strain ATCC 13281. The genomic DNA was digested tocompletion with BamHI and EcoRI. Fragments having an average size ofabout 4 kbp were isolated from an agarose gel, ligated to plasmidpLitmus28 (New England Biolabs, Beverly, Mass.), which was then digestedwith BamHI and EcoRI. The resulting DNA was transformed intoultracompetent E. coli TOP10F′ (Invitrogen, Carlsbad, Calif.). Thetransformants were plated on MacConkey agar plates containing 1% sucroseand 100 μg/mL ampicillin and screened for purple colonies. Plasmid DNAwas isolated from the purple transformants and sequenced using primersM13 Forward (SEQ ID NO:45), M13 Reverse (SEQ ID NO:46), scr1 (SEQ IDNO:160), scr2 (SEQ ID NO:161), scr3 (SEQ ID NO:162), and scr4 (SEQ IDNO:163). The plasmid containing cscB, cscK, and cscA (cscBKA) genes wasdesignated pScr1.

Plasmid pScrI was digested with XhoI and treated with the Klenowfragment of DNA polymerase to make blunt ends. The plasmid was thendigested with AgeI, and the 4,179 bp cscBKA gene cluster fragment wasgel-purified. Plasmid pBHR-Ptrc-ald(opt) was prepared as described inExample 17 and was digested with AgeI and NaeI. The resulting 6,003 bppBHR-Ptrc-ald(opt) fragment was gel-purified. The cscBKA fragment wasligated with the pBHR-Ptrc-ald(opt), yielding pBHR-Ptrc-ald(opt)-cscAKB.Plasmid pBHR-Ptrc-ald(opt)-cscAKB was transformed into E. coli NovaXGelectrocompetent cells (Novagen, Madison, Wis.) and sucrose utilizationwas confirmed by plating the transformants on McConkey agar platescontaining 2% sucrose and 25 μg/mL kanamycin. In thepBHR-Ptrc-ald(opt)-cscAKB construct, the sucrose utilization genes werecloned downstream of Ptrc-ald(opt) as a separate fragment in the ordercscA, cscK, and cscB.

Alternatively, the sucrose utilization genes were cloned in the oppositedirection in pBHR-Ptrc-ald(opt). Plasmid pBHR-Ptrc-ald(opt) was digestedwith ScaI and AgeI, and the 5,971 bp pBHR-Ptrc-ald(opt) fragment wasgel-purified. The 4,179 bp cscBKA fragment, prepared as described above,was ligated with the pBHR-Ptrc-ald(opt) fragment, yieldingpBHR-Ptrc-ald(opt)-cscBKA. Plasmid pBHR-Ptrc-ald(opt)-cscBKA wastransformed into E. coli NovaXG electrocompetent cells (Novagen,Madison, Wis.) and sucrose utilization was confirmed by plating thetransformants on McConkey agar plates containing 2% sucrose and 25 μg/mLkanamycin. In the pBHR-Ptrc-ald(opt)-cscBKA construct, the sucroseutilization genes were cloned as a separate fragment downstream ofPtrc-ald(opt) in the order cscB, cscK, and cscA.

Demonstration of 1-Butanol Production from Glucose or Sucrose UsingRecombinant E. coli.

E. coli strain MG1655 1.5GI-yqhD::Cm (described in Example 17) wastransformed with plasmids pTrc99a-E-C-H-T (prepared as described inExample 17) and pBHR-Ptrc-ald(opt)-cscAKB or pBHR-Ptrc-ald(opt)-cscBKAto produce two strains, MG1655 1.5GI-yqhD::Cm/pTrc99a-E-C-H-T/pBHR-Ptrc-ald(opt)-cscAKB #9 and MG1655 1.5GI-yqhD::Cm/pTrc99a-E-C-H-T/pBHR-Ptrc-ald(opt)-cscBKA #1. Starter culturesof the two strains were prepared by growing the cells in LB mediumcontaining 25 μg/mL of kanamycin and 100 μg/mL of carbenicillin. Thesecells were then used to inoculate shake flasks (approximately 175 mLtotal volume) containing 50, 70 and 150 mL of TM3a/glucose medium (withappropriate antibiotics) to represent high, medium and low oxygenconditions, respectively, as described in Example 17. A third strain, E.coli MG1655/pScrI, grown in TM3a/glucose medium containing 100 μg/mLcarbenicillin, was used as a negative control. For each of the strains,an identical set of flasks was prepared with TM3a/sucrose medium (withappropriate antibiotics). TM3a/sucrose medium is identical toTM3a/glucose medium except that sucrose (10 g/L) replaces glucose. Theflasks were inoculated at a starting OD₅₅₀ of ≦0.03 units and incubatedas described in Example 17. With the exception of the negative controlflasks, IPTG was added to the flasks (final concentration of 0.04 mM)when the cultures reached an OD₅₅₀ between 0.2 and 1.8 units. The cellswere harvested when the OD₅₅₀ of the cultures increased at least 3-fold.

Approximately 24 h after inoculation, an aliquot of the broth wasanalyzed by HPLC (Shodex Sugar SH1011 column) with refractive index (R1)detection and GC (HP-INNOWax column, 30 m×0.53 mm id, 1 μm filmthickness) with flame ionization detection (FID) for 1-butanol content,as described in the General Methods section.

The concentrations of 1-butanol in cultures following growth in theglucose and sucrose-containing media are given in Table 20 and Table 21,respectively. Both recombinant E. coli strains containing the 1-butanolbiosynthetic pathway produced 1-butanol from glucose and sucrose underall oxygen conditions, while the negative control strain produced nodetectable 1-butanol.

TABLE 20 Production of 1-butanol from glucose by recombinant E. colistrains MG1655 1.5GI-yqhD::Cm/pTrc99a-E-C- H-T/pBHR-Ptrc-ald(opt)-cscAKB#9 and MG1655 1.5GI- yqhD::Cm/pTrc99a-E-C-H-T/pBHR-Ptrc-ald(opt)-cscBKA#1 Strain O₂ Level 1-butanol, mM molar yield, % cscBKA #1 high 0.01 0.03cscBKA #1 medium 0.20 0.43 cscBKA #1 low 0.07 0.21 cscAKB #9 high 0.010.02 cscAKB #9 medium 0.17 0.35 cscAKB #9 low 0.04 0.12 pScr1 high Notdetected Not detected pScr1 medium Not detected Not detected pScrl lowNot detected Not detected

TABLE 21 Production of 1-butanol from sucrose by recombinant E. colistrains. Strain O₂ Level 1-butanol, mM molar yield, % cscBKA #1 high0.02 0.10 cscBKA #1 medium 0.02 0.11 cscBKA #1 low 0.01 0.09 cscAKB #9high 0.03 0.11 cscAKB #9 medium 0.03 0.15 cscAKB #9 low 0.02 0.10 pScr1high Not detected Not detected pScr1 medium Not detected Not detectedpScr1 low Not detected Not detected

Example 20 Production of 1-Butanol from Sucrose Using Recombinant B.Subtilis

This example describes the production of 1-butanol from sucrose usingrecombinant Bacillus subtilis. Two independent isolates of B. subtilisstrain ΔsacB::T-H-C::erm #28/pHT01-ald-EB (Example 18) were examined for1-butanol production essentially as described in Example 18. The strainswere inoculated into shake flasks (approximately 175 mL total volume)containing either 20 mL or 100 mL of medium to simulate high and lowoxygen conditions, respectively. Medium A was exactly as described inExample 18, except that glucose was replaced with 5 g/L of sucrose.Medium B was identical to the TM3a/glucose medium described in Example17, except that glucose was replaced with 10 g/L sucrose and the mediumwas supplemented with (per L) 10 mL of a 5 g/L solution of each ofL-methionine, L-tryptophan, and L-lysine. The flasks were inoculated ata starting OD₅₅₀ of ≦0.1 units, capped with vented caps, and incubatedat 34° C. with shaking at 300 rpm.

Approximately 24 h after inoculation, an aliquot of the broth wasanalyzed by GC (HP-INNOWax column, 30 m×0.53 mm id, 1.0 μm filmthickness) with FID detection for 1-butanol content, as described in theGeneral Methods section. The results of the 1-butanol determinations aregiven in Table 22. The recombinant Bacillus strain containing the1-butanol biosynthetic pathway produced detectable levels of 1-butanolunder high and low oxygen conditions in both media.

TABLE 22 Production of 1-butanol from sucrose by B. subtilis strainΔsacB::T-H-C::erm #28/pHT01-ald-EB Strain Medium O₂ Level 1-BuOH,mM^(1,2) none A Not applicable Not detected pHT01-ald-EB a A high +pHT01-ald-EB b A high + pHT01-ald-EB a A low 0.01 pHT01-ald-EB b A low0.01 none B Not applicable Not detected pHT01-ald-EB a B high +pHT01-ald-EB b B high + pHT01-ald-EB a B low 0.04 pHT01-ald-EB b B low0.03 ¹Concentration determined by GC. ²“+” indicates qualitativepresence of 1-butanol. Strain suffixes “a” and “b” indicate separateisolates.

Example 21 Production of 1-Butanol from Glucose and Sucrose UsingRecombinant Saccharomyces cerevisiae

This Example describes the production of 1-butanol in the yeastSaccharomyces cerevisiae. Of the six genes encoding enzymes catalyzingthe steps in the 1-butanol biosynthetic pathway, five were cloned intothree compatible yeast 2 micron (2μ) plasmids and co-expressed inSaccharomyces cerevisiae. The “upper pathway” is defined as the firstthree enzymatic steps, catalyzed by acetyl-CoA acetyltransferase (thIA,thiolase), 3-hydroxybutyryl-CoA dehydrogenase (hbd), and crotonase(crt). The lower pathway is defined as the fourth (butyl-CoAdehydrogenase, ter) and the fifth (butylaldehyde dehydrogenase, ald)enzymatic steps of the pathway. The last enzymatic step of the 1-butanolpathway is catalyzed by alcohol dehydrogenase, which may be encoded byendogenous yeast genes, e.g., adhIO and adhII.

Expression of genes in yeast typically requires a promoter, followed bythe gene of interest, and a transcriptional terminator. A number ofconstitutive yeast promoters were used in constructing expressioncassettes for genes encoding the 1-butanol biosynthetic pathway,including FBA, GPD, and GPM promoters. Some inducible promoters, e.g.GAL1, GAL10, CUP1 were also used in intermediate plasmid construction,but not in the final demonstration strain. Several transcriptionalterminators were used, including FBAt, GPDt, GPMt, ERG10t, and GAL1t.Genes encoding the 1-butanol biosynthetic pathway were first subclonedinto a yeast plasmid flanked by a promoter and a terminator, whichyielded expression cassettes for each gene. Expression cassettes wereoptionally combined in a single vector by gap repair cloning, asdescribed below. For example, the three gene cassettes encoding theupper pathway were subcloned into a yeast 2μ plasmid. The ter and aldgenes were each expressed individually in the 2μ plasmids.Co-transformation of all three plasmids in a single yeast strainresulted in a functional 1-butanol biosynthetic pathway. Alternatively,several DNA fragments encoding promoters, genes, and terminators weredirectly combined in a single vector by gap repair cloning.

Methods for Constructing Plasmids and Strains in Yeast Saccharomycescerevisiae.

Basic yeast molecular biology protocols including transformation, cellgrowth, gene expression, gap repair recombination, etc. are described inMethods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecularand Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink(Eds.), Elsevier Academic Press, San Diego, Calif.).

The plasmids used in this Example were E. coli-S. cerevisiae shuttlevectors, pRS423, pRS424, pRS425, and pRS426 (American Type CultureCollection, Rockville, Md.), which contain an E. coli replication origin(e.g., pMB1), a yeast 2μ origin of replication, and a marker fornutritional selection. The selection markers for these four vectors areHis3 (vector pRS423), Trp1 (vector pRS424), Leu2 (vector pRS425) andUra3 (vector pRS426). These vectors allow strain propagation in both E.coli and yeast strains. A yeast haploid strain BY4741 (MA Ta his3Δ1leu2Δ0 met15Δ0 ura3Δ0) (Research Genetics, Huntsville, Ala., alsoavailable from ATCC 201388) and a diploid strain BY4743 (MATa/alphahis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0/ura3Δ0)(Research Genetics, Huntsville, Ala., also available from ATCC 201390)were used as hosts for gene cloning and expression. Construction ofexpression vectors for genes encoding 1-butanol biosynthetic pathwayenzymes were performed by either standard molecular cloning techniquesin E. coli or by the gap repair recombination method in yeast.

The gap repair cloning approach takes advantage of the highly efficienthomologous recombination in yeast. Typically, a yeast vector DNA isdigested (e.g., in its multiple cloning site) to create a “gap” in itssequence. A number of insert DNAs of interest are generated that containa ≧21 bp sequence at both the 5′ and the 3′ ends that sequentiallyoverlap with each other, and with the 5′ and 3′ terminus of the vectorDNA. For example, to construct a yeast expression vector for “Gene X”, ayeast promoter and a yeast terminator are selected for the expressioncassette. The promoter and terminator are amplified from the yeastgenomic DNA, and Gene X is either PCR amplified from its source organismor obtained from a cloning vector comprising Gene X sequence. There isat least a 21 bp overlapping sequence between the 5′ end of thelinearized vector and the promoter sequence, between the promoter andGene X, between Gene X and the terminator sequence, and between theterminator and the 3′ end of the linearized vector. The “gapped” vectorand the insert DNAs are then co-transformed into a yeast strain andplated on the SD minimal dropout medium, and colonies are selected forgrowth of cultures and mini preps for plasmid DNAs. The presence ofcorrect insert combinations can be confirmed by PCR mapping. The plasmidDNA isolated from yeast (usually low in concentration) can then betransformed into an E. coli strain, e.g. TOP10, followed by mini prepsand restriction mapping to further verify the plasmid construct. Finallythe construct can be verified by sequence analysis. Yeast transformantsof positive plasmids are grown in SD medium for performing enzyme assaysto characterize the activities of the enzymes expressed by the genes ofinterest.

Yeast cultures were grown in YPD complex medium or Synthetic Minimaldropout medium containing glucose (SD medium) and the appropriatecompound mixtures that allow complementation of the nutritionalselection markers on the plasmids (Methods in Enzymology, Volume 194,Guide to Yeast Genetics and Molecular and Cell Biology, 2004, Part A,pp. 13-15). The sugar component in the SD drop out medium was 2%glucose. For 1-Butanol production, yeast cultures were also grown inSynthetic Minimal dropout medium with 2% sucrose (SS medium).

For enzyme activity analysis, a single colony of each strain wasstreaked onto a fresh plate containing SD minimal drop out medium andincubated at 30° C. for 2 days. The cells on this plate were used toinoculate 20 mL of SD drop out medium and in a 125 mL shake flask andgrown overnight at 30° C., with shaking at 250 rpm. The optical density(OD₆₀₀) of the overnight culture was measured, and the culture wasdiluted to an OD₆₀₀=0.1 in 250 mL of the same medium in a 1.0 L shakeflask, and grown at 30° C. with shaking at 250 rpm to an OD₆₀₀ ofbetween 0.8 to 1.0. The cells were then harvested by centrifugation at2000×g for 10 min, and resuspended in 20 mL of 50 mM Tris-HCl buffer, pH8.5. Enzyme assays were performed as described above.

Construction of Plasmid pNY102 for thIA and hbd Co-Expression.

A number of dual expression vectors were constructed for theco-expression of thIA and hbd genes. The Saccharomyces cerevisiae ERG10gene is a functional ortholog of the thIA gene. Initially, a dual vectorof ERG10 and hbd was constructed using the yeast GAL1-GAL10 divergentdual promoter, the GAL1 terminator (GAL1t) and the ERG10 terminator(ERG10t). The ERG10 gene-ERG10t DNA fragment was PCR amplified fromgenomic DNA of Saccharomyces cerevisiae strain BY4743, using primersOT731 (SEQ ID NO:164) and OT732 (SEQ ID NO:165). The yeast GAL1-GAL10divergent promoter was also amplified by PCR from BY4743 genomic DNAusing primers OT733 (SEQ ID NO:166) and OT734 (SEQ ID NO:167). The hbdgene was amplified from E. coli plasmid pTrc99a-E-C-H-T (described inExample 17) using PCR primers OT735 (SEQ ID NO:168) and OT736 (SEQ IDNO:169). GAL1t was amplified from BY4743 genomic DNA using primers OT737(SEQ ID NO:170) and OT738 (SEQ ID NO:171). Four PCR fragments,erg10-ERG10t, GAL1-GAL10 promoters, hbd, and GAL1t, were thus obtainedwith approximately 25 bp overlapping sequences between each adjacent PCRfragment. GAL1t and ERG10-ERG10t fragments each contain approximately 25bp overlapping sequences with the yeast vector pRS425. To assemble thesesequences by gap repair recombination, the DNA fragments wereco-transformed into the yeast strain BY4741 together with vector pRS425which was digested with BamHI and HindIII enzymes. Colonies wereselected from SD-Leu minimal plates, and clones with inserts wereidentified by PCR amplification. The new plasmid was named pNY6(pRS425.ERG10t-erg10-GAL10-GAL1-hbd-GAL1t). Further confirmation wasperformed by restriction mapping.

The yeast strain BY4741 (pNY6), prepared by transforming plasmid pNY6into S. cerevisiae BY4741, showed good Hbd activity but no thiolaseactivity. Due to the lack of thiolase activity, the ERG10 gene wasreplaced with the thIA gene by gap repair recombination. The thIA genewas amplified from E. coli vector pTrc99a-E-C-H-T by PCR using primersOT797 (SEQ ID NO:172) which adds a SphI restriction site, and OT798 (SEQID NO:173) which adds an AscI restriction site. Plasmid pNY6 wasdigested with SphI and PstI restriction enzymes, gel-purified, andco-transformed into yeast BY4741 along with the PCR product of thIA. Dueto the 30 bp overlapping sequences between the PCR product of thIA andthe digested pNY6, the thIA gene was recombined into pNY6 between theGAL10 promoter and the ERG10t terminator. This yielded plasmid pNY7(pRS425.ERG10t-thIA-GAL10-GAL1-hbd-GAL1t), which was verified by PCR andrestriction mapping.

In a subsequent cloning step based on gap repair recombination, theGAL10 promoter in pNY7 was replaced with the CUP1 promoter, and the GAL1promoter was replaced with the strong GPD promoter. This plasmid, pNY10(pRS425. ERG10t-thIA-CUP1-GPD-hbd-GAL1t) allows for the expression ofthe thIA gene under CUP1, a copper inducible promoter, and theexpression of the hbd gene under the GPD promoter. The CUP1 promotersequence was PCR amplified from yeast BY4743 genomic DNA using primersOT806 (SEQ ID NO:174), and OT807 (SEQ ID NO:175). The GPD promoter wasamplified from BY4743 genomic DNA using primers OT808 (SEQ ID NO:176)and OT809 (SEQ ID NO:177). PCR products of the CUP1 and the GPDpromoters were combined with pNY7 plasmid digested with NcoI and SphIrestriction enzymes. From this gap repair cloning step, plasmid pNY10was constructed, which was verified by PCR and restriction mapping.Yeast BY4741 strain containing pNY10 had Hbd activity, but no ThIAactivity. The Hbd activity under GPD promoter was significantly improvedcompared to the GAL1 promoter controlled Hbd activity (1.8 U/mg vs. 0.40U/mg). Sequencing analysis revealed that the thIA gene in pNY10 had aone base deletion near the 3′ end, which resulted in a truncatedprotein. This explains the lack of thiolase activity in the strain.

Plasmid pNY12 was constructed with the correct thIA gene sequence. ThethIA gene was cut from the vector pTrc99a-E-C-H-T by digestion with SphIand AscI. The FBA1 promoter was PCR amplified from BY4743 genomic DNAusing primers OT799 (SEQ ID NO:178) and OT761 (SEQ ID NO:179), anddigested with SalI and SphI restriction enzymes. The thIA gene fragmentand FBA1 promoter fragment were ligated into plasmid pNY10 at AscI andSalI sites, generating plasmid pNY12 (pRS425.ERG10t-thIA-FBA1), whichwas confirmed by restriction mapping. pNY12 was transformed into yeaststrain BY4741 and the resulting transformant showed a ThIA activity of1.66 U/mg.

The FBA1 promoter-th/A gene fragment from pNY12 was re-subcloned intopNY10. The pNY10 vector was cut with the AscI restriction enzyme andligated with the AscI digested FBA1 promoter-th/A gene fragment isolatedfrom plasmid pNY12. This created a new plasmid with two possible insertorientations. The clones with FBA1 and GPD promoters located adjacent toeach other in opposite orientation were chosen and this plasmid wasnamed pNY102. pNY102 (pRS425. ERG10t-thIA-FBA1-GPD-hbd-GAL1t) wasverified by restriction mapping. Strain DPD5206 was made by transformingpNY102 into yeast strain BY4741. The ThIA activity of DPD5206 was 1.24U/mg and the Hbd activity was 0.76 U/mg.

Construction of Plasmid pNY11 for crt Expression.

The crt gene expression cassette was constructed by combining the GPM1promoter, the crt gene, and the GPM1t terminator into vector pRS426using gap repair recombination in yeast. The GPM1 promoter was PCRamplified from yeast BY4743 genomic DNA using primers OT803 (SEQ IDNO:180) and OT804 (SEQ ID NO:181). The crt gene was amplified using PCRprimers OT785 (SEQ ID NO:182) and OT786 (SEQ ID NO:183) from E. coliplasmid pTrc99a-E-C-H-T. The GPM1t terminator was PCR amplified fromyeast BY4743 genomic DNA using OT787 (SEQ ID NO:184) and OT805 (SEQ IDNO:185). Yeast vector pRS426 was digested with BamHI and HindIII and wasgel-purified. This DNA was co-transformed with the PCR products of theGPM1 promoter, the crt gene and the GPM1 terminator into yeast BY4741competent cells. Clones with the correct inserts were verified by PCRand restriction mapping and the resulting yeast strain BY4741 (pNY11:pRS426-GPM1-crt-GPM1t) had a Crt activity of 85 U/mg.

Construction of Plasmid pNY103 for thIA, hbd and crt Co-Expression.

For the co-expression of the upper 1-butanol pathway enzymes, the crtgene cassette from pNY11 was subcloned into plasmid pNY102 to create anhbd, thIA, and crt expression vector. A 2,347 bp DNA fragment containingthe GPM1 promoter, the crt gene, and the GPM1 terminator was cut fromplasmid pNY11 with SacI and NotI restriction enzymes and cloned intovector pNY102, which was digested with NotI and partially digested withSacI, producing the expression vector pNY103 (pRS425.ERG10t-thIA-FBA1-GPD-hbd-GAL1t-GPM1t-crt-GPM1). Following confirmationof the presence of all three cassettes in pNY103 bp digestion withHindIII, the plasmid was transformed into yeast BY4743 cells and thetransformed yeast strain was named DPD5200. When grown under standardconditions, DPD5200 showed ThIA, Hbd, and Crt enzyme activities of 0.49U/mg, 0.21 U/mg and 23.0 U/mg, respectively.

Construction of Plasmid pNY8 for ald Expression.

A codon optimized gene named tery (SEQ ID NO:186), encoding the Terprotein (SEQ ID NO:187), and a codon optimized gene named aldy (SEQ IDNO:188), encoding the Ald protein (SEQ ID NO:189) were synthesized usingpreferred codons of Saccharomyces cerevisiae. Plasmid pTERy containingthe codon optimized ter gene and pALDy containing the codon optimizedald gene were made by DNA2.0 (Palo Alto, Calif.).

To assemble pNY8 (pRS426.GPD-ald-GPDt), three insert fragments includinga PCR product of the GPD promoter (synthesized from primers OT800 (SEQID NO:190) and OT758, (SEQ ID NO:191), and BY4743 genomic DNA), an aldygene fragment excised from pALDy by digestion with NcoI and SfiI (SEQ IDNO:188), and a PCR product of the GPD terminator (synthesized fromprimers OT754 (SEQ ID NO:192) and OT755 (SEQ ID NO:193), and BY4743genomic DNA) were recombined with the BamHI, HindIII digested pRS426vector via gap repair recombination cloning. Yeast BY4741 transformationclones were analyzed by PCR mapping. The new plasmid thus constructed,pNY8, was further confirmed by restriction mapping. The yeast BY4741transformants containing pNY8 were analyzed for Ald activity and thespecific activity towards butyryl-CoA was approximately 0.07 U/mg.

Construction of Plasmids pNY9 and pNY13 for ter Expression.

The codon optimized tery gene was cloned into vector pRS426 undercontrol of the FBA1 promoter by gap repair cloning. The FBA1 promoterwas PCR amplified from yeast BY4743 genomic DNA using primers OT760 (SEQID NO:194) and OT792 (SEQ ID NO:195). The tery gene was obtained bydigestion of plasmid pTERy by SphI and NotI restriction enzymes thatresulted in the fragment given as SEQ ID NO:186. The PCR fragment ofFBA1 terminator was generated by PCR from yeast BY4743 genomic DNA usingprimers OT791 (SEQ ID NO:196) and OT765 (SEQ ID NO:197). Three DNAfragments, the FBA1 promoter, the ter gene and the FBA1 terminator, werecombined with the BamHI, HindIII digested pRS426 vector and transformedinto yeast BY4741 bp gap repair recombination. The resulting plasmid,pNY9 (pRS426-FBA1-tery-FBA1t) was confirmed by PCR mapping, as well asrestriction digestion. The yeast BY4741 transformant of pNY9 produced aTer activity of 0.26 U/mg.

To make the final 1-butanol biosynthetic pathway strain, it wasnecessary to construct a yeast expression strain that contained severalplasmids, each with a unique nutritional selection marker. Since theparent vector pRS426 contained a Ura selection marker, the terexpression cassette was subcloned into vector pRS423, which contained aHis3 marker. A 3.2 kb fragment containing the FBA1-tery-FBA1t cassettewas isolated from plasmid pNY9 bp digestion with SacI and XhoIrestriction enzymes, and ligated into vector pRS423 that was cut withthese same two enzymes. The new plasmid, pNY13 (pRS423-FBA1-tery-FBA1t)was mapped by restriction digestion. pNY13 was transformed into BY4741strain and the transformant was cultured in SD-His medium, yielding astrain with a Ter activity of 0.19 U/mg.

Construction of a Yeast Strain Containing 1-Butanol Biosynthetic PathwayGenes for Demonstration of 1-Butanol Production.

As described above, yeast strain DPD5200 was constructed bytransformation of plasmid pNY103 into S. cerevisiae strain BY4743, whichallows co-expression of thIA, hbd and crt genes. Yeast competent cellsof DPD5200 were prepared as described above, and plasmids pNY8 and pNY13were co-transformed into DPD5200, generating strain DPD5213. DPD5213allows for the simultaneous constitutive expression of five genes in the1-butanol biosynthetic pathway, thIA, hbd, crt, ter and aid. StrainDPD5212 (S. cerevisiae strain BY4743 transformed with empty plasmids,pRS425 and pRS426) was used as a negative control. Four independentisolates of strain DPD5213 were grown on SD-Ura,-Leu,-His dropoutminimal medium in the presence of either 2% glucose or 2% sucrose toallow the growth complementation of all three plasmids. A single isolateof DPD5212 was similarly grown in appropriate medium.

To demonstrate 1-butanol production by aerobic cultures, a single colonyof each strain was streaked onto a fresh agar plate containing SDminimal drop out growth medium (containing 2% glucose) or SS minimaldrop out growth medium (containing 2% sucrose) and incubated at 30° C.for 2 days. Cells from these plates were used to inoculate 20 mL of theminimal drop out medium (either SD or SS) in 125 mL plastic shake flasksand were grown overnight at 30° C. with shaking at 250 rpm. The opticaldensity (OD₆₀₀) of the overnight culture was measured, the culture wasdiluted to OD₆₀₀ of 0.1 in 25 mL of the same medium in a 125 mL shakeflask, and grown at 30° C. with shaking at 250 rpm.

Aliquots of the culture were removed at 24 h and 48 h for GC analysis of1-butanol production (HP-INNOWax column, 30 m×0.53 mm id, 1 μm filmthickness) with FID detection, as described in the General Methodssection. The results of the GC analysis are given in Table 23.

TABLE 23 Production of 1-butanol from glucose and sucrose by S.cerevisiae strain DPD5213 1-butanol at 24 h, 1-butanol at 48 h, Strain¹Sugar mg/L² mg/L² DPD5212 Glucose Not detected Not detected DPD5213 aGlucose 0.4 0.5 DPD5213 b Glucose 0.9 0.2 DPD5213 c Glucose 1.0 0.6DPD5213 d Glucose 0.8 0.3 DPD5212 Sucrose Not detected Not detectedDPD5213 a Sucrose Not detected 1.7 DPD5213 b Sucrose Not detected 1.3DPD5213 c Sucrose 0.2 1.5 DPD5213 d Sucrose 0.6 0.9 ¹Independentisolates are indicated by a-d. ²Concentration determined by GC.

Example 22 Prophetic Expression of the 1-Butanol Biosynthetic Pathway inLactobacillus Plantarum

The purpose of this prophetic Example is to describe how to express the1-butanol biosynthetic pathway in Lactobacillus plantarum. The six genesof the 1-butanol pathway, encoding six enzyme activities, are dividedinto two operons for expression. The first three genes of the pathway(thI, hbd, and crt, encoding the enzymes acetyl-CoA acetyltransferase,3-hydroxybutyryl-CoA dehydrogenase, and crotonase, respectively) areintegrated into the chromosome of Lactobacillus plantarum by homologousrecombination using the method described by Hols et al. (Appl. Environ.Microbiol. 60:1401-1413 (1994)). The last three genes (EgTER, aid, andbdhB, encoding the enzymes butyryl-CoA dehydrogenase, butyraldehydedehydrogenase and butanol dehydrogenase, respectively) are cloned intoan expression plasmid and transformed into the Lactobacillus straincarrying the integrated upper pathway 1-butanol genes. Lactobacillus isgrown in MRS medium (Difco Laboratories, Detroit, Mich.) at 37° C.Chromosomal DNA is isolated from Lactobacillus plantarum as described byMoreira et al. (BMC Microbiol. 5:15 (2005)).

Integration.

The thI-hbd-crt cassette under the control of the synthetic P11 promoter(Rud et al., Microbiology 152:1011-1019 (2006)) is integrated into thechromosome of Lactobacillus plantarum ATCC BAA-793 (NCIMB 8826) at theldhL1 locus by homologous recombination. To build the ldhL integrationtargeting vector, a DNA fragment from Lactobacillus plantarum (GenbankNC_(—)004567) with homology to ldhL is PCR amplified with primers LDHEcoRV F (SEQ ID NO:198) and LDH AatIIR (SEQ ID NO:199). The 1986 bp PCRfragment is cloned into pCR4Blunt-TOPO and sequenced. ThepCR4Blunt-TOPO-ldhL1 clone is digested with EcoRV and AatII releasing a1982 bp ldhL1 fragment that is gel-purified. The integration vectorpFP988, described in Example 18, is digested with HindIII and treatedwith Klenow DNA polymerase to blunt the ends. The linearized plasmid isthen digested with AatII and the 2931 by vector fragment isgel-purified. The EcoRV/AatII ldhL1 fragment is ligated with the pFP988vector fragment and transformed into E. coli Top10 cells. Transformantsare selected on LB agar plates containing ampicillin (100 μg/mL) and arescreened by colony PCR to confirm construction of pFP988-ldhL.

To add a selectable marker to the integrating DNA, the Cm gene with itspromoter is PCR amplified from pC194 (Genbank NC_(—)002013) with primersCm F (SEQ ID NO:200) and Cm R (SEQ ID NO:201), amplifying a 836 bp PCRproduct. The amplicon is cloned into pCR4Blunt-TOPO and transformed intoE. coli Top10 cells, creating pCR4Blunt-TOPO-Cm. After sequencing toconfirm that no errors are introduced by PCR, the Cm cassette isdigested from pCR4Blunt-TOPO-Cm as an 828 bp MluI/SwaI fragment and isgel-purified. The ldhL-homology containing integration vectorpFP988-ldhL is digested with MluI and SwaI and the 4740 bp vectorfragment is gel-purified. The Cm cassette fragment is ligated with thepFP988-ldhL vector creating pFP988-DldhL::Cm.

Finally the thI-hbd-crt cassette from pFP988Dss-T-H-C, described inExample 18, is modified to replace the amylase promoter with thesynthetic P11 promoter. Then, the whole operon is moved intopFP988-DldhL::Cm. The P11 promoter is built by oligonucleotide annealingwith primer P11 F (SEQ ID NO:202) and P11 R (SEQ ID NO:203). Theannealed oligonucleotide is gel-purified on a 6% Ultra PAGE gel (EmbiTec, San Diego, Calif.). The plasmid pFP988Dss-T-H-C is digested withXhoI and SmaI and the 9 kbp vector fragment is gel-purified. Theisolated P11 fragment is ligated with the digested pFP988Dss-T-H-C tocreate pFP988-P11-T-H-C. Plasmid pFP988-P11-T-H-C is digested with XhoIand BamHI and the 3034 bp P11-T-H-C fragment is gel-purified.pFP988-DldhL::Cm is digested with XhoI and BamHI and the 5558 bp vectorfragment isolated. The upper pathway operon is ligated with theintegration vector to create pFP988-DldhL-P11-THC::Cm.

Integration of pFP988-DldhL-P11-THC::Cm into L. plantarum BAA-793 toform L. plantarum ΔldhL1::T-H-C::Cm Comprising Exogenous thI, hbd, andcrt Genes.

Electrocompetent cells of L. plantarum are prepared as described byAukrust, T. W., et al. (In: Electroporation Protocols forMicroorganisms; Nickoloff, J. A., Ed.; Methods in Molecular Biology,Vol. 47; Humana Press, Inc., Totowa, N.J., 1995, pp 201-208). Afterelectroporation, cells are outgrown in MRSSM medium (MRS mediumsupplemented with 0.5 M sucrose and 0.1 M MgCl₂) as described by Aukrustet al. supra for 2 h at 37° C. without shaking. Electroporated cells areplated for selection on MRS plates containing chloramphenicol (10 μg/mL)and incubated at 37° C. Transformants are initially screened by colonyPCR amplification to confirm integration, and initial positive clonesare then more rigorously screened by PCR amplification with a battery ofprimers.

Plasmid Expression of EgTER, ald, and bdhB Genes.

The three remaining 1-butanol genes are expressed from plasmid pTRKH3(O'Sullivan D J and Klaenhammer T R, Gene 137:227-231 (1993)) under thecontrol of the L. plantarum ldhL promoter (Ferain et al., J. Bacteriol.176:596-601 (1994)). The ldhL promoter is PCR amplified from the genomeof L. plantarum ATCC BAA-793 with primers PldhL F (SEQ ID NO:204) andPldhL R (SEQ ID NO:205). The 369 bp PCR product is cloned intopCR4Blunt-TOPO and sequenced. The resulting plasmid,pCR4Blunt-TOPO-PldhL is digested with SacI and BamHI releasing the 359bp PldhL fragment.

pHT01-ald-EB, described in Example 18, is digested with SacI and BamHIHand the 10503 bp vector fragment is recovered by gel purification. ThePldhL fragment and vector are ligated creating pHT01-PldhL-ald-EB.

To subclone the ldhL promoter-ald-EgTER-bdh cassette, pHT01-PldhL-ald-EBis digested with MluI and the ends are treated with Klenow DNApolymerase. The linearized vector is digested with Sail and the 4270 bpfragment containing the PldhL-AEB fragment is gel-purified. PlasmidpTRKH3 is digested with Sail and EcoRV and the gel-purified vectorfragment is ligated with the PldhL-AEB fragment. The ligation mixture istransformed into E. coli Top 10 cells and transformants are plated onBrain Heart Infusion (BHI, Difco Laboratories, Detroit, Mich.) platescontaining erythromycin (150 mg/L). Transformants are screened by PCR toconfirm construction of pTRKH3-ald-E-B. The expression plasmid,pTRKH3-ald-E-B is transformed into L. plantarum ΔldhL1::T-H-C::Cm byelectroporation, as described above.

L. plantarum ΔldhL1::T-H-C::Cm containing pTRKH3-ald-E-B is inoculatedinto a 250 mL shake flask containing 50 mL of MRS medium pluserythromycin (10 μg/mL) and grown at 37° C. for 18 to 24 h withoutshaking. After 18 h to 24, 1-butanol is detected by HPLC or GC analysis,as described in the General Methods section.

Example 23 Prophetic Expression of the 1-Butanol Biosynthetic Pathway inEnterococcus faecalis

The purpose of this prophetic Example is to describe how to express the1-butanol biosynthetic pathway in Enterococcus faecalis. The completegenome sequence of Enterococcus faecalis strain V583, which is used asthe host strain for the expression of the 1-butanol biosynthetic pathwayin this Example, has been published (Paulsen et al., Science299:2071-2074 (2003)). Plasmid pTRKH3 (O'Sullivan D J and Klaenhammer TR, Gene 137:227-231 (1993)), an E. coli/Gram-positive shuttle vector, isused for expression of the six genes (thIA, hbd, crt, EgTER, aid, bdhB)of the 1-butanol pathway in one operon. pTRKH3 contains an E. coliplasmid p15A replication origin and the pAMβ1 replicon, and twoantibiotic resistance selection markers, tetracycline resistance anderythromycin resistance. Tetracycline resistance is only expressed in E.coli, and erythromycin resistance is expressed in both E. coli andGram-positive bacteria. Plasmid pAMf31 derivatives can replicate in E.faecalis (Poyart et al., FEMS Microbiol. Lett. 156:193-198 (1997)). Theinducible nisA promoter (PnisA), which has been used for efficientcontrol of gene expression by nisin in a variety of Gram-positivebacteria including Enterococcus faecalis (Eichenbaum et al., Appl.Environ. Microbiol. 64:2763-2769 (1998)), is used to control expressionof the six desired genes encoding the enzymes of the 1-butanolbiosynthetic pathway.

The linear DNA fragment (215 bp) containing the nisA promoter(Chandrapati et al., Mol. Microbiol. 46(2):467-477 (2002)) isPCR-amplified from Lactococcus lactis genomic DNA with primersF-PnisA(EcoRV) (SEQ ID NO:206) and R-PnisA(PmeI BamHI) (SEQ ID NO:207).The 215 bp PCR fragment is digested with EcoRV and BamHI, and theresulting PnisA fragment is gel-purified. Plasmid pTRKH3 is digestedwith EcoRV and BamHI and the vector fragment is gel-purified. Thelinearised pTRKH3 is ligated with the PnisA fragment. The ligationmixture is transformed into E. coli Top10 cells by electroporation andtransformants are selected following overnight growth at 37° C. on LBagar plates containing erythromycin (25 μg/mL). The transformants arethen screened by colony PCR with primers F-PnisA(EcoRV) andR-PnisA(BamHI) to confirm the correct clone of pTRKH3-PnisA.

Plasmid pTRKH3-PnisA is digested with PmeI and BamHI, and the vector isgel-purified. Plasmid pHTO1-ald-EgTER-bdhB is constructed as describedin Example 18 and is digested with SmaI and BamHI, and the 2,973 bpald-EgTER-bdhB fragment is gel-purified. The 2,973 bp ald-EgTER-bdhBfragment is ligated into the pTRKH3-PnisA vector at the PmeI and BamHIsites. The ligation mixture is transformed into E. coli Top10 cells byelectroporation and transformants are selected following incubation at37° C. overnight on LB agar plates containing erythromycin (25 μg/mL).The transformants are then screened by colony PCR with primers aldforward primer N27F1 (SEQ ID NO: 31) and bdhB reverse primer N65 (SEQ IDNO: 44). The resulting plasmid is named pTRKH3-PnisA-ald-EgTER-bdhB(=pTRKH3-A-E-B).

Plasmid pTRKH3-A-E-B is purified from the transformant and used forfurther cloning of the remaining genes (thIA, hbd, crt) into the BamHIsite located downstream of the bdhB gene. Plasmid pTRKH3-A-E-B isdigested with BamHI and treated with the Klenow fragment of DNApolymerase to make blunt ends. Plasmid pFP988Dss-thIA-hbd-crt(=pFP988Dss-T-H-C) is constructed as described in Example 18 and isdigested with SmaI and BamHI. The resulting 2,973 bp th/A-hbd-crtfragment is treated with the Klenow fragment of DNA polymerase to makeblunt ends and is gel-purified. The 2,973 bp th/A-hbd-crt fragment isligated with the linearised pTRKH3-A-E-B. The ligation mixture istransformed into E. coli Top10 cells by electroporation andtransformants are selected following overnight growth at 37° C. on LBagar plates containing erythromycin (25 μg/mL). The transformants arethen screened by colony PCR with primers thIA forward primer N7 (SEQ IDNO: 21) and crt reverse primer N4 (SEQ ID NO: 18). The resulting plasmidis named pTRKH3-PnisA-ald-EgTER-bdhB-thIA-hbd-crt (=pTRKH3-A-E-B-T-H-C).Plasmid pTRKH3-A-E-B-T-H-C is prepared from the E. coli transformantsand transformed into electro-competent E. faecalis V583 cells byelectroporation using methods known in the art (Aukrust, T. W., et al.In: Electroporation Protocols for Microorganisms; Nickoloff, J. A., Ed.;Methods in Molecular Biology, Vol. 47; Humana Press, Inc., Totowa, N.J.,1995, pp 217-226), resulting in E. faecalis V583/pTRKH3-A-E-B-T-H-C.

The second plasmid containing nisA regulatory genes, nisR and nisK, theadd9 spectinomycin resistance gene, and the pSH71 origin of replicationis transformed into E. faecalis V583/pTRKH3-A-E-B-T-H-C byelectroporation. The plasmid containing pSH71 origin of replication iscompatible with pAMβ1 derivatives in E. faecalis (Eichenbaum et al.,supra). Double drug resistant transformants are selected on LB agarplates containing erythromycin (25 μg/mL) and spectinomycin (100 μg/mL).

The resulting E. faecalis strain V5838 harboring two plasmids, i.e., anexpression plasmid (pTRKH3-A-E-B-T-H-C) and a regulatory plasmid(pSH71-nisRK), is inoculated into a 250 mL shake flask containing 50 mLof Todd-Hewitt broth supplemented with yeast extract (0.2%) (Fischettiet al., J. Exp. Med. 161:1384-1401 (1985)), nisin (20 μg/mL) (Eichenbaumet al., supra), erythromycin (25 μg/mL), and spectinomycin (100 μg/mL).The flask is incubated without shaking at 37° C. for 18 to 24 h, afterwhich time, 1-butanol production is measured by HPLC or GC analysis, asdescribed in the General Methods section.

Example 24 Increased Tolerance of Saccharomyces cerevisiae to 1-Butanolat Decreased Growth Temperatures

Tolerance levels were determined for yeast strain Saccharomycescerevisiae BY4741 (described in Example 21) at 25° C. and 30° C. asfollows. The strain was cultured in YPD medium. Overnight cultures inthe absence of any test compound were started in 25 mL of YPD medium in150 mL flasks with incubation at 30° C. or at 25° C. in shaking waterbaths. The next morning, each overnight culture was diluted into a 500mL flask-containing 300 mL of fresh medium to an initial OD600 of about0.1. The flasks were incubated in shaking water baths at 30° C. or 25°C., using the same temperature as used for each overnight culture. Thelarge cultures were incubated for 3 hours and then were split intoflasks in the absence (control) and in the presence of 1% or 2% of1-butanol. Growth was followed by measuring OD600 for six hours afteraddition of the 1-butanol. The ΔOD₆₀₀ was calculated by subtracting theinitial OD₆₀₀ from the final OD₆₀₀ at 6 hours. The percent growthinhibition relative to the control culture was calculated as follows: %Growth Inhibition=100−[100(Sample ΔOD₆₀₀/Control ΔOD₆₀₀)]. The resultsare summarized in Table 24 below and indicate that growth of strainBY4741 was less inhibited by 1% 1-butanol at 25° C. than by 1% 1-butanolat 30° C.

TABLE 24 Growth of Saccharomyces cerevisiae Strain BY4741 at 25° C. and30° C. with 1-Butanol. % 1-Butanol Temperature ° C. % Growth Inhibition1 30 64 1 25 43 2 30 No growth 2 25 No growth

1-27. (canceled)
 28. A recombinant microbial host cell comprising DNAmolecules encoding polypeptides that catalyze each of the followingsubstrate to product conversions: i) acetyl-CoA to acetoacetyl-CoA; ii)acetoacetyl-CoA to 3-hydroxybutyryl-CoA; iii) 3-hydroxybutyryl-CoA tocrotonyl-CoA; iv) crotonyl-CoA to butyryl-CoA; v) butyryl-CoA tobutyraldehyde; and vi) butyraldehyde to 1-butanol; wherein thepolypeptide that catalyzes the substrate to product conversion ofacetyl-CoA to acetoacetyl-CoA is acetyl-CoA acetyltransferase; thepolypeptide that catalyzes the substrate to product conversion ofacetoacetyl-CoA to 3-hydroxybutyryl-CoA is 3-hydroxybutyryl-CoAdehydrogenase; the polypeptide that catalyzes the substrate to productconversion of 3-hydroxybutyryl-CoA to crotonyl-CoA is crotonase; thepolypeptide that catalyzes the substrate to product conversion ofcrotonyl-CoA to butyryl-CoA is butyryl-CoA dehydrogenase; thepolypeptide that catalyzes the substrate to product conversion ofbutyryl-CoA to butyraldehyde is butyraldehyde dehydrogenase; and thepolypeptide that catalyzes the substrate to product conversion ofbutyraldehyde to 1-butanol is butanol dehydrogenase.
 29. The recombinantmicrobial host cell of claim 28, wherein the acetyl-CoAacetyltransferase has an amino acid sequence having at least 95%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 129, SEQ ID NO: 131, and SEQ IDNO: 133 based on the Clustal W method of alignment using the defaultparameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250series of protein weight matrix.
 30. The recombinant microbial host cellof claim 28, wherein the 3-hydroxybutyryl-CoA dehydrogenase has an aminoacid sequence having at least 95% identity to an amino acid sequenceselected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 135, SEQID NO: 137, and SEQ ID NO: 139 based on the Clustal W method ofalignment using the default parameters of GAP PENALTY=10, GAP LENGTHPENALTY=0.1, and Gonnet 250 series of protein weight matrix.
 31. Therecombinant microbial host cell of claim 28, wherein the crotonase hasan amino acid sequence having at least 95% identity to an amino acidsequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO:141, SEQ ID NO: 143, and SEQ ID NO: 145 based on the Clustal W method ofalignment using the default parameters of GAP PENALTY=10, GAP LENGTHPENALTY=0.1, and Gonnet 250 series of protein weight matrix
 32. Therecombinant microbial host cell of claim 28, wherein the butyryl-CoAdehydrogenase has an amino acid sequence having at least 95% identity toan amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, and SEQ ID NO: 187based on the Clustal W method of alignment using the default parametersof GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series ofprotein weight matrix.
 33. The recombinant microbial host cell of claim28, wherein the butyraldehyde dehydrogenase has an amino acid sequencehaving at least 95% identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 12, SEQ ID NO: 153, and SEQ ID NO: 189based on the Clustal W method of alignment using the default parametersof GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series ofprotein weight matrix.
 34. The recombinant microbial host cell of claim28, wherein the butanol dehydrogenase has an amino acid sequence havingat least 95% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 153, SEQ ID NO:155, and SEQ ID NO: 157 based on the Clustal W method of alignment usingthe default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, andGonnet 250 series of protein weight matrix.
 35. The recombinantmicrobial host cell of claim 28, wherein the acetyl-CoAacetyltransferase has an amino acid sequence having at least 95%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 129, SEQ ID NO: 131, and SEQ IDNO: 133 based on the Clustal W method of alignment using the defaultparameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250series of protein weight matrix; wherein the 3-hydroxybutyryl-CoAdehydrogenase has an amino acid sequence having at least 95% identity toan amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO: 135, SEQ ID NO: 137, and SEQ ID NO: 139 based on theClustal W method of alignment using the default parameters of GAPPENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of proteinweight matrix; wherein the crotonase has an amino acid sequence havingat least 95% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 141, SEQ ID NO: 143, and SEQ IDNO: 145 based on the Clustal W method of alignment using the defaultparameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250series of protein weight matrix; wherein the butyryl-CoA dehydrogenasehas an amino acid sequence having at least 95% identity to an amino acidsequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO:147, SEQ ID NO: 149, SEQ ID NO: 151, and SEQ ID NO: 187 based on theClustal W method of alignment using the default parameters of GAPPENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of proteinweight matrix; wherein the butyraldehyde dehydrogenase has an amino acidsequence having at least 95% identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 12, SEQ ID NO: 153, and SEQ IDNO: 189 based on the Clustal W method of alignment using the defaultparameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250series of protein weight matrix; and wherein the butanol dehydrogenasehas an amino acid sequence having at least 95% identity to an amino acidsequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 153, SEQ ID NO: 155, and SEQ ID NO: 157 based on theClustal W method of alignment using the default parameters of GAPPENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of proteinweight matrix.
 36. The recombinant microbial host cell of claim 28,wherein the recombinant microbial host cell is selected from the groupconsisting of Clostridium, Zymomonas, Escherichia, Salmonella,Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus,Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Saccharomyces, Pichia, Candida, and Hansenula.
 37. Therecombinant microbial host cell of claim 36, wherein the recombinantmicrobial host cell is selected from the group consisting of Escherichiacoli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillusmacerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillusplantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcusfaecalis, Bacillus subtilis, and Saccharomyces cerevisiae.
 38. A methodto reduce the sensitivity of a recombinant microbial host cell to1-butanol comprising: a) providing a recombinant microbial host cellwhich produces 1-butanol, wherein the recombinant microbial host cellcomprises DNA molecules encoding polypeptides that catalyze each of thefollowing substrate to product conversions: i) acetyl-CoA toacetoacetyl-CoA; ii) acetoacetyl-CoA to 3-hydroxybutyryl-CoA; iii)3-hydroxybutyryl-CoA to crotonyl-CoA; iv) crotonyl-CoA to butyryl-CoA;v) butyryl-CoA to butyraldehyde; and vi) butyraldehyde to 1-butanol;wherein at least one of the DNA molecules is heterologous to therecombinant microbial host cell; b) growing the recombinant microbialhost cell in a fermentation culture; and c) determining the metabolicactivity of the fermentation culture by monitoring one or more metabolicparameters selected from optical density, pH, respiratory quotient,fermentable carbon substrate utilization, CO₂ production, and 1-butanolproduction.
 39. The method of claim 38, further comprising the stepadjusting the one or more metabolic parameters to support the metabolicactivity.
 40. The method of claim 39, wherein a decrease in one or moreof the metabolic parameters indicates a decrease in metabolic activity.41. The method of claim 40, wherein the adjusting the one or moremetabolic parameters is lowering the temperature of the fermentationculture when a decrease in metabolic activity is detected.
 42. Themethod of claim 38, wherein the polypeptide that catalyzes the substrateto product conversion of acetyl-CoA to acetoacetyl-CoA is acetyl-CoAacetyltransferase; the polypeptide that catalyzes the substrate toproduct conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA is3-hydroxybutyryl-CoA dehydrogenase; the polypeptide that catalyzes thesubstrate to product conversion of 3-hydroxybutyryl-CoA to crotonyl-CoAis crotonase; the polypeptide that catalyzes the substrate to productconversion of crotonyl-CoA to butyryl-CoA is butyryl-CoA dehydrogenase;the polypeptide that catalyzes the substrate to product conversion ofbutyryl-CoA to butyraldehyde is butyraldehyde dehydrogenase; and thepolypeptide that catalyzes the substrate to product conversion ofbutyraldehyde to 1-butanol is butanol dehydrogenase.